Figure 3 ( A ) Confocal immunofluorescence of muscle biopsy from Pt 01 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( which recognizes protein fragment encoded by exons 26 - 30 , green signal ) and anti laminin - 2 ( to delineate muscle fibers , red signal ) ; dapi identifies nuclei ( blue signal ) . In the left image only anti dystrophin staining is shown . No dystrophin positive fibers were observed . ( B ) Confocal immunofluorescence of muscle biopsy from Pt 02 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( green signal ) and anti lamininin - 2 ( red signal ) ; dapi identifies nuclei ( blue signal ) . In the left image only anti dystrophin staining is shown . Some fiber shows mild and discontinuous dystrophin staining . ( C ) Immunofluorescence of muscle biopsy from Pt 05 taken before ( left , gastrocnemius ) and after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody ( which recognizes exon 77 - 79 ) . The number and intensity of dystrophin - positive fibers is increased in the post - treatment biopsy . ( D ) Immunofluorescence of muscle biopsy from Pt 06 taken before ( left , gastrocnemius ) and after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody . Few fibers show scattered dystrophin staining , without obvious differences between pre and post - treatment samples . ( E ) Results of western blot analysis involving dystrophin antibodies dys1 , Mandys18 and Manex46e ( recognizing respectively exon 26 - 30 , 17 - 35 , and 46 ) , of total protein extracts ( 20 µg ) obtained from post - treatment biopsy specimens of Pt 01 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . No bands corresponding to full length dystrophin were observed . A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot . ( F ) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e , of total protein extracts ( 10 - 20 µg ) obtained from pre - treatment ( performed at time of diagnosis ) and post - treatment biopsy specimens of Pt 02 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . One faint band corresponding to full length dystrophin is observed only in the post - treatment samples with both Mandys18 and Manex46e antibodies . A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot . ( G ) Results of western blot analysis involving dystrophin antibodies Mandys106 ( recognizing exon 43 ) and dys1 , of total protein extracts ( 80 µg , following protein concentration by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment and post - treatment biopsy specimens of Pt 05 ( gastrocnemius ) ; Ct was used as positive dystrophin control , CtDmd was used as dystrophin negative control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full length dystrophin are observed in pre and post - treatment samples with both antibodies However , bands in post - treatment sample appeared higher in amount . A schematic representation of the dystrophin point mutation ( black vertical bar ) and the region recognized by used antibodies is depicted above the western blot . ( H ) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1 , of total protein extracts ( 80 µg , following protein concentration by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment and post - treatment biopsy specimens of Pt 06 ( gastrocnemius ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full length dystrophin are observed in pre and post - treatment samples with both antibodies Bands in pre - treatment sample appeared higher in amount as compared to post - treatment sample . A schematic representation of the dystrophin point mutation ( black vertical bar ) and the region recognized by used antibodies is depicted above the western blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26-30, green signal) and anti laminin-2 (to delineate muscle fibers, red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. No dystrophin positive fibers were observed.(B) Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (green signal) and anti lamininin-2 (red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining.(C) Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody (which recognizes exon 77-79). The number and intensity of dystrophin-positive fibers is increased in the post-treatment biopsy.(D) Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre and post-treatment samples.(E) Results of western blot analysis involving dystrophin antibodies dys1, Mandys18 and Manex46e (recognizing respectively exon 26-30, 17-35, and 46), of total protein extracts (20 µg) obtained from post-treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. No bands corresponding to full length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(F) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10-20 µg) obtained from pre-treatment (performed at time of diagnosis) and post-treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. One faint band corresponding to full length dystrophin is observed only in the post-treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(G) Results of western blot analysis involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as positive dystrophin control, CtDmd was used as dystrophin negative control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies However, bands in post-treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.(H) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies Bands in pre-treatment sample appeared higher in amount as compared to post-treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.


Figure 4 ( A ) Axial T1 - weighted images of the right thighs obtained in patients immediately before ( upper panel ) and 18 months after MAB infusion . Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected . ( B ) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients . SIR ( left image ) and MVI ( right image ) of the quadriceps . Red line : Pt 01 ; dotted green line : Pt 02 ; dotted purple line : Pt 03 ; dotted dark blue line : Pt 05 ; dotted light blue line : Pt 06 ) . Empty dots correspond to pre - treatment measures . Orange dots correspond to post - transplantation measurement . The black line corresponds to the median , while gray dotted lines to 75 ° , 90 ° and 95 ° percentile . Please note the modification of the trend of Pt 05 after treatment . ( C ) Representative quantile spline regressions of post - transplant trend amelioration of MRI quantitative parameters in Pt 05 . Upper left : SIR of the quadriceps . Lower left : SIR of thigh flexor muscles . Upper right : MVI of the quadriceps . Lower right : MVI of the semitendinosus muscles . Please note the significant modification of the patient trend ( red lines ) compared to median measurements obtained with 85 MRI examinations of untreated patients . Empty dots correspond to pre - treatment measures . Full dots correspond to post - transplantation measurement . The grey dotted line corresponds to the time of the first intra - arterial transplantation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Axial T1-weighted images of the right thighs obtained in patients immediately before (upper panel) and 18 months after MAB infusion. Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected. (B) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients. SIR (left image) and MVI (right image) of the quadriceps. Red line: Pt 01; dotted green line: Pt 02; dotted purple line: Pt 03; dotted dark blue line: Pt 05; dotted light blue line: Pt 06). Empty dots correspond to pre-treatment measures. Orange dots correspond to post-transplantation measurement. The black line corresponds to the median, while gray dotted lines to 75°, 90° and 95° percentile. Please note the modification of the trend of Pt 05 after treatment.(C) Representative quantile spline regressions of post-transplant trend amelioration of MRI quantitative parameters in Pt 05. Upper left: SIR of the quadriceps. Lower left: SIR of thigh flexor muscles. Upper right: MVI of the quadriceps. Lower right: MVI of the semitendinosus muscles. Please note the significant modification of the patient trend (red lines) compared to median measurements obtained with 85 MRI examinations of untreated patients. Empty dots correspond to pre-treatment measures. Full dots correspond to post-transplantation measurement. The grey dotted line corresponds to the time of the first intra-arterial transplantation.


Figure 5 ( A ) North Star scale measurement ( NSAA score ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions ; Pt05 showed stabilization even in the subsequent period , whereas Pt02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial . ( B ) Six minute walking test ( meters ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions ; Pt05 showed stabilization even in the subsequent period , whereas Pt 02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial . ( C ) Time to run 10 meters ( time ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions ; Pt 05 showed stabilization even in the subsequent period , whereas Pt 02 showed progressive increase of time until lost of ambulation . Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) North Star scale measurement (NSAA score) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.(B) Six minute walking test (meters) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.(C) Time to run 10 meters (time) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions; Pt 05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive increase of time until lost of ambulation. Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial.


Figure 6Panel A : viral and donor specific T - cell responses were measured by IFN - γELISpot at different time points : before the beginning of treatment ( PRE ) , prior to each infusion and 2 months after the fourth infusion . Infusions are indicated by arrows . Patient ' s peripheral blood mononuclear cells ( PBMC ) were challenged with irradiated autologous lymphoblastoid cell lines ( EBV line host ) , with cytomegalovirus glycin extract ( CMV antigen ) ( Gehrz et al , 1987 ) and with the following irradiated cells harvested from the donor : untreated mesoangioblasts ( MABdonor ) , MAB activated by 48 hour - exposure to 500 IU / mL IFN - γ ( γMABdonor ) , myotubes differentiated from MAB ( myotubesdonor ) and PBMC ( PBMCdonor ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . Donor PBMC were challenged with autologous targets as negative controls . Results are expressed as number of specific cells / 105PBMC and calculated according to the following formula : number of spots produced by patient ' s PBMC - number of spots produced by donor ' s PBMC . Results for patientsPt 01 , Pt 02 , Pt 03 , Pt 05 and Pt 06 are shown . Panel B : dystrophin specific T - cell responses were measured by IFN - γ ELISpot at two time points : before the beginning of treatment ( PRE , white bar ) and 2 months after the fourth infusion ( black bar ) . PBMC were challenged with a library of 15 - mer overlapping peptides covering the protein sequence ( pool 1 - 8 ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . The grey area represents the threshold calculated on a cohort of healthy donors . Results for Pt 01 , Pt 02 , Pt 03 , Pt 05 and Pt 06 are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Panel A: viral and donor specific T-cell responses were measured by IFN-γELISpot at different time points: before the beginning of treatment (PRE), prior to each infusion and 2 months after the fourth infusion. Infusions are indicated by arrows. Patient's peripheral blood mononuclear cells (PBMC) were challenged with irradiated autologous lymphoblastoid cell lines (EBV line host), with cytomegalovirus glycin extract (CMV antigen)(Gehrz et al, 1987) and with the following irradiated cells harvested from the donor: untreated mesoangioblasts (MABdonor), MAB activated by 48 hour-exposure to 500 IU/mL IFN-γ (γMABdonor), myotubes differentiated from MAB (myotubesdonor) and PBMC (PBMCdonor). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. Donor PBMC were challenged with autologous targets as negative controls. Results are expressed as number of specific cells/105PBMC and calculated according to the following formula: number of spots produced by patient's PBMC - number of spots produced by donor's PBMC. Results for patientsPt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.Panel B: dystrophin specific T-cell responses were measured by IFN-γ ELISpot at two time points: before the beginning of treatment (PRE, white bar) and 2 months after the fourth infusion (black bar). PBMC were challenged with a library of 15-mer overlapping peptides covering the protein sequence (pool 1-8). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. The grey area represents the threshold calculated on a cohort of healthy donors. Results for Pt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.


Figure 1 ( A ) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain , and lower in the adult brain . Data are presented as mean ± SEM . ( A ) ng PTX3 / mg of total proteins , E15 = 5 , 702 ± 0 . 365 , P0 = 4 . 904 ± 0 . 865 , P7 = 3 . 818 ± 0 . 530 , P14 = 2 . 140 ± 0 . 107 , P28 = 3 . 125 ± 0 . 525 , P90 = 2 . 212 ± 0 . 324 . One - way ANOVA analysis of variance followed by post hoc Tukey test : E15 vs P14 * * p = 0 . 001 , E15 vs P28 * p = 0 . 022 , E15 vs P90 * * p = 0 . 002 ; P0 vs P14 $ p = 0 . 013 , P0 vs P90 $ p = 0 . 016 ; 4 animals for each time point ( B ) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain , and lower in the adult brain . Data are presented as mean ± SEM ( B ) qPCR fold change normalized on E15 , E15 = 1 . 037 ± 0 . 154 , P0 = 0 . 711 ± 0 . 094 , P7 = 0 . 472 ± 0 . 0524 , P14 = 0 . 135 ± 0 . 007 , P28 = 0 . 168 ± 0 . 020 , P90 = 0 . 183 ± 0 . 029 . One - way ANOVA analysis of variance followed by post hoc Tukey test : E15 vs P7 * * * p = 0 . 0001 , E15 vs P14 * * * * p < 0 . 0001 , E15 vs P2 * * * * p < 0 . 0001 , E15 vs P90 * * * * p < 0 . 0001 ; P0 vs P14 $ $ $ p = 0 . 0007 , P0 vs P28 $ $ p = 0 . 001 , P0 vs P90 $ $ p = 0 . 002 ; 4 animals for each time pointQuantitation of PTX3 levels in pure astrocyte and neuronal cultures . ( C ) ELISA assay performed on astrocyte and neuronal culture mediu Only astrocytes release PTX3 ( ELISA , astro = 3 . 699 ± 1 . 129 , neu = N . D . , not detectable . 3 independent astrocyte cultures evaluated Data are presented as mean ± SEMQuantitation of PTX3 levels in pure astrocyte and neuronal cultures ( D ) rea time qPCR performed on astrocyte and neuronal lysates qPCR astro = 1 . 167 ± 0 . 288 ; neu = 0 . 148 ± 0 . 057 ; Mann - Whitney test * * p = 0 . 0095 . 2 independent neuronal cultures evaluated . Data are presented as mean ±

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM. (A) ng PTX3/mg of total proteins, E15=5,702±0.365, P0=4.904±0.865, P7=3.818±0.530, P14=2.140±0.107, P28=3.125±0.525, P90=2.212±0.324. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P14 **p=0.001, E15 vs P28 *p=0.022, E15 vs P90 **p=0.002; P0 vs P14 $p=0.013, P0 vs P90 $p=0.016 ; 4 animals for each time point(B) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM (B) qPCR fold change normalized on E15, E15=1.037±0.154, P0=0.711±0.094, P7=0.472±0.0524, P14=0.135±0.007, P28=0.168±0.020, P90=0.183±0.029. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P7 ***p=0.0001, E15 vs P14 **** p<0.0001, E15 vs P2 ****p<0.0001, E15 vs P90 ****p<0.0001; P0 vs P14 $$$p=0.0007, P0 vs P28 $$p=0.001, P0 vs P90 $$p=0.002; 4 animals for each time pointQuantitation of PTX3 levels in pure astrocyte and neuronal cultures. (C) ELISA assay performed on astrocyte and neuronal culture mediu Only astrocytes release PTX3 (ELISA, astro=3.699±1.129, neu=N.D., not detectable. 3 independent astrocyte cultures evaluated Data are presented as mean±SEMQuantitation of PTX3 levels in pure astrocyte and neuronal cultures (D) rea time qPCR performed on astrocyte and neuronal lysates qPCR astro=1.167±0.288; neu=0.148±0.057; Mann-Whitney test **p=0.0095. 2 independent neuronal cultures evaluated. Data are presented as mean±SEM


Figure 2A ) Representative mEPSC traces recorded from control and PTX3 - treated ( 1µg / ml ; 48hrs ) neurons . B ) mEPSC frequency quantitation ( Hz , Ctr = 0 . 618 ± 0 . 069 ; PTX3 = 1 . 991 ± 0 . 313 ; Number of neurons : Ctr = 22 , PTX3 = 16 ; 3 independent experiments , Mann - Whitney test . Data are presented as a distribution plus mean ± SEM ) . C ) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes ( pA , Ctr = 12 . 76 ± 0 . 813 ; PTX3 = 16 . 07 ± 0 . 709 . Number of neurons : Ctr = 22 , PTX3 = 16 ; 3 independent experiments , Mann - Whitney test . Data are presented as a distribution plus mean ± SEM . Cumulative probability distributions are analyzed by Kolmogorov - Smirnov testD ) Representative images showing 14DIV neurons stained for surface AMPAR ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Inset : Example of surface synaptic AMPARs cluster ( GluA & Bsn ) . Scale bar : 5 µm . E ) Quantification of the surface synaptic AMPARs ( GluA & Bsn ) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure ( Ctr = 1 ± 0 . 051 , TTX = 1 . 512 ± 0 . 080 , PTX3 = 1 . 294 ± 0 . 081 , PTX3 heat - inactivated = 0 . 959 ± 0 . 044 ; number of fields examined : 27 , 36 , 37 , 22 , 19 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 , followed by post hoc Tukey test for multiple comparison as indicated in figure ; 3 independent experiments , data are presented as normalized mean values ± SEM ) . F ) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content ( Ctr : 1 ± 0 . 065 , PTX3 : 1 . 023 ± 0 . 068 ; number of fields : Ctr = 26 , PTX3 = 26 ; unpaired t - test , 3 independent experiments , data are presented as normalized mean values ± SEM ) G ) Representative mEPSC traces recorded from control , C - term PTX3 fragment or N - term PTX3 fragment - treated cultures . H ) mEPSC frequency quantitation ( Hz , Ctr = 0 . 668 ± 0 . 078 ; C - term = 0 . 523 ± 0 . 056 ; N - term = 1 . 228 ± 0 . 121 . Number of neurons : Ctr = 23 , C - term = 10 , N - term = 23 ; 3 independent experiments . Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated , data are presented as a distribution plus mean ± SEM ) . I ) mEPSC amplitude quantitation ( pA , Ctr = 16 . 81 ± 0 . 78 ; C - term = 15 . 79 ± 1 . 37 ; N - term = 19 . 78 ± 0 . 87 . Number of neurons : Ctr = 23 , C - term = 10 , N - term = 23 ; 3 independent experiments . One - way ANOVA analysis of variance , p = 0 . 013 followed by post hoc Tukey test as indicated in figure , data are presented as a distribution plus mean ± SEM ) and cumulative probability distribution of mEPSC amplitudes ( Kolmogorov - SmirnovJ ) Representative images of 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . K ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N - terminal peptide application , but not upon C - terminal peptide exposure . TTX is used as positive control ( Ctr = 1 . 000 ± 0 . 066 . TTX = 1 . 395 ± 0 . 113 , C - term = 0 . 717 ± 0 . 047 , N - term = 1 . 349 ± 0 . 069 . Number of fields examined : 55 , 31 , 18 , 54 , respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± SEM ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Representative mEPSC traces recorded from control and PTX3-treated (1µg/ml; 48hrs) neurons. B) mEPSC frequency quantitation (Hz, Ctr=0.618±0.069; PTX3=1.991±0.313; Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM). C) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr=12.76±0.813; PTX3=16.07±0.709. Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM. Cumulative probability distributions are analyzed by Kolmogorov-Smirnov testD) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA&Bsn). Scale bar: 5 µm. E) Quantification of the surface synaptic AMPARs (GluA&Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr=1±0.051, TTX=1.512±0.080, PTX3=1.294±0.081, PTX3 heat-inactivated=0.959±0.044; number of fields examined: 27, 36, 37, 22, 19 respectively; one-way ANOVA analysis of variance, p<0.0001, followed by post hoc Tukey test for multiple comparison as indicated in figure; 3 independent experiments, data are presented as normalized mean values±SEM). F) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content (Ctr: 1±0.065, PTX3: 1.023±0.068; number of fields: Ctr=26, PTX3=26; unpaired t-test, 3 independent experiments, data are presented as normalized mean values±SEM)G) Representative mEPSC traces recorded from control, C-term PTX3 fragment or N-term PTX3 fragment-treated cultures. H) mEPSC frequency quantitation (Hz, Ctr=0.668±0.078; C-term=0.523±0.056; N-term=1.228±0.121. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated, data are presented as a distribution plus mean±SEM). I) mEPSC amplitude quantitation (pA, Ctr=16.81±0.78; C-term=15.79±1.37; N-term=19.78±0.87. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. One-way ANOVA analysis of variance, p=0.013 followed by post hoc Tukey test as indicated in figure, data are presented as a distribution plus mean±SEM) and cumulative probability distribution of mEPSC amplitudes (Kolmogorov-SmirnovJ) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. K) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N-terminal peptide application, but not upon C-terminal peptide exposure. TTX is used as positive control (Ctr=1.000±0.066. TTX=1.395±0.113, C-term=0.717±0.047, N-term=1.349±0.069. Number of fields examined: 55, 31, 18, 54, respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


Figure 3A ) Low and high magnification images of control and PTX3 - treated neurons stained for the PNN main component , aggrecan ( red ) , the synaptic proteins PSD95 ( green ) and Bsn ( blue ) . Scale bar : 5µmB - D ) PTX3 application induces a remodeling of the PNN in culture , as assessed by the increase mean intensity and integrated density value of the synapse - co - localizing aggrecan signal whereas no difference in the total area of aggrecan is evident ( integrated density : Ctr = 1 ± 0 . 103 , PTX3 = 1 . 874 ± 0 . 197 ; mean intensity : Ctr = 1 . 000 ± 0 . 088 , PTX3 = 1 . 556 ± 0 . 107 ; total area : Ctr = 1 ± 0 . 179 , PTX3 = 0 . 982 ± 0 . 149 . Number of fields examined : 26 Ctr , 22 PTX3 ; Mann - Whitney test ; 3 independent experiments , data are presented as normalized mean values ± SEM ) E ) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan ( red ) , DAPI ( cyan ) and βIII - tubulin ( green ) and confocal analysis . Scale bar : 20µmF ) Representative images showing 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µmG ) HAse treatment blocks PTX3 - induced synaptic surface AMPA receptors clustering ( Ctr = 1 . 000 ± 0 . 075 , PTX3 = 1 . 425 ± 0 . 088 , PTX3 + HAse = 0 . 961 ± 0 . 053 , HAse = 1 . 080 ± 0 . 087 . Number of fields examined : 37 , 23 , 53 , 28 respectively ; Kruskall - Wallis test , p = 0 . 0004 followed by post hoc Tukey test as indicated in figure ; 3 independent experiments , data are presented as normalized mean value ± SEM ) H ) Representative images showing 14DIV WT and TSG6 KO neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µmI ) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures . On the contrary WT cultures ( from littermates ) display increased surface GluA & Bsn / Bsn upon PTX3 treatment . A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures ( WT = 1 ± 0 . 06 ; WT + PTX3 = 1 . 389 ± 0 . 113 ; WT + TTX = 1 . 698 ± 0 . 109 ; Number of fields examined : 40 , 39 , 32 respectively ; Kruskall - Wallis test p < 0 . 0001 followed by post hoc Dunn " s test . TSG6 KO = 1 . 000 ± 0 . 047 ; TSG6 KO + PTX3 = 0 . 979 ± 0 . 048 ; TSG6 KO + TTX = 1 . 363 ± 0 . 077 . Number of fields examined : 54 , 57 , 29 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure . n = 3 independent experiments , data are presented as normalized mean valuesJ ) Representative mEPSC traces recorded from WT and TSG6 KO littermates cultures treated or not with PTX3 ( 1μg / ml for 48hrs ) . K ) On the contrary of WT cultures , mEPSC frequency quantitation shows no increase in frequency in TSG6 KO cultures when treated with PTX3 ( Normalized Frequency , WT Ctr : 1 . 000 ± 0 . 074 ; Wt + PTX3 = 1 . 912 ± 0 . 289 ; TSG6 KO Ctr = 1 ± 0 . 079 ; TSG6 KO + PTX3 = 1 . 226 ± 0 . 096 ; 22 , 20 , 30 , 24 cells respectively , 6 TSG6 KO mice and 4 WT littermates . One way ANOVA test p = 0 . 002 followed by post hoc Dunn ' s test , data are presented as normalized mean ± SEM plus the distribution ) . L ) Cumulative probability plot of mEPSC amplitude and average mEPSC amplitude quantitation ( inset ) showing no difference in amplitude ( WT Ctr : 1 . 000 ± 0 . 034 , Wt + PTX3 = 1 . 069 ± 0 . 046 , TSG6 KO Ctr = 1 . 000 ± 0 . 028 ; TSG6 KO + PTX3 = 1 . 074 ± 0 . 67 ; 22 , 20 , 30 , 24 cells respectively , 6 TSG6 KO mice and 4 WT littermates . One way ANOVA test p = 0 . 817 followed by post hoc Dunn ' s test , data are presented as normalized mean ± SEM plus the distribution . Cumulative distribution is analyzed by Kolmogorov - Smirnov test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Low and high magnification images of control and PTX3-treated neurons stained for the PNN main component, aggrecan (red), the synaptic proteins PSD95 (green) and Bsn (blue). Scale bar: 5µmB-D) PTX3 application induces a remodeling of the PNN in culture, as assessed by the increase mean intensity and integrated density value of the synapse-co-localizing aggrecan signal whereas no difference in the total area of aggrecan is evident (integrated density: Ctr=1±0.103, PTX3=1.874±0.197; mean intensity: Ctr=1.000±0.088, PTX3=1.556±0.107; total area: Ctr=1±0.179, PTX3=0.982±0.149. Number of fields examined: 26 Ctr, 22 PTX3; Mann-Whitney test; 3 independent experiments, data are presented as normalized mean values±SEM)E) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan (red), DAPI (cyan) and βIII-tubulin (green) and confocal analysis. Scale bar: 20µmF) Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µmG) HAse treatment blocks PTX3-induced synaptic surface AMPA receptors clustering (Ctr=1.000±0.075, PTX3=1.425±0.088, PTX3+HAse=0.961±0.053, HAse=1.080±0.087. Number of fields examined: 37, 23, 53, 28 respectively; Kruskall-Wallis test, p=0.0004 followed by post hoc Tukey test as indicated in figure; 3 independent experiments, data are presented as normalized mean value±SEM)H) Representative images showing 14DIV WT and TSG6 KO neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µmI) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures. On the contrary WT cultures (from littermates) display increased surface GluA&Bsn/Bsn upon PTX3 treatment. A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures (WT=1±0.06; WT+PTX3=1.389±0.113; WT+TTX=1.698±0.109; Number of fields examined: 40, 39, 32 respectively; Kruskall-Wallis test p<0.0001 followed by post hoc Dunn"s test. TSG6 KO=1.000±0.047; TSG6 KO+PTX3=0.979±0.048; TSG6 KO+TTX=1.363±0.077. Number of fields examined: 54, 57, 29 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. n=3 independent experiments, data are presented as normalized mean valuesJ) Representative mEPSC traces recorded from WT and TSG6 KO littermates cultures treated or not with PTX3 (1μg/ml for 48hrs). K) On the contrary of WT cultures, mEPSC frequency quantitation shows no increase in frequency in TSG6 KO cultures when treated with PTX3(Normalized Frequency, WT Ctr: 1.000±0.074; Wt+PTX3=1.912±0.289; TSG6 KO Ctr=1±0.079; TSG6 KO+PTX3=1.226±0.096; 22, 20, 30, 24 cells respectively, 6 TSG6 KO mice and 4 WT littermates. One way ANOVA test p=0.002 followed by post hoc Dunn's test, data are presented as normalized mean±SEM plus the distribution). L) Cumulative probability plot of mEPSC amplitude and average mEPSC amplitude quantitation (inset) showing no difference in amplitude (WT Ctr: 1.000±0.034, Wt+PTX3=1.069±0.046, TSG6 KO Ctr =1.000±0.028; TSG6 KO+PTX3=1.074±0.67; 22, 20, 30, 24 cells respectively, 6 TSG6 KO mice and 4 WT littermates. One way ANOVA test p=0.817 followed by post hoc Dunn's test, data are presented as normalized mean±SEM plus the distribution. Cumulative distribution is analyzed by Kolmogorov-Smirnov test).


Figure 4A ) Representative images showing 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µmB ) Blocking β1 integrins activity by using the specific anti - β1 Integrin monoclonal antibody prevents the PTX3 - induced postsynaptic AMPARs recruitment ( Ctr = 1 . 000 ± 0 . 074 , PTX3 = 1 . 504 ± 0 . 098 , αCD29 + PTX3 = 0 . 766 ± 0 . 097 , αCD29 = 0 . 810 ± 0 . 108 . Number of fields examined : 37 , 33 , 23 , 24 respectively ; one way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test ; 3 independent experiments , data are presented as normalized mean values ± SEM ) C ) Western blotting analysis of p - ERK levels on lysates from control and PTX3 - treated neurons upon 30 minutes stimulation ( Ctr = 1 ± 0 ; PTX3 = 1 . 453 ± 0 . 195 , 6 independent experiments , unpaired t - test , data are presented normalized on control and as mean ± SEM ) D ) Representative mEPSC traces recorded from the indicated experimental conditions . E ) mEPSC frequency quantitation showing that pre - incubation with PD98059 ( 30μM ) completely prevents the PTX3 - dependent increase of mEPSC frequency ( Hz , Ctr = 0 . 911 ± 0 . 104 ; PTX3 = 1 . 748 ± 0 . 273 ; PTX3 + PD = 0 . 518 ± 0 . 055 ; PD = 0 . 491 ± 0 . 104 . Number of neurons : Ctr = 14 , PTX3 = 14 , PTX3 + PD = 13 ; PD = 9 ; 3 independent experiments . One - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure . Data are presented as a distribution plus mean ± SEM ) . F ) inset : average mEPSC amplitude quantitation ( pA ) , Ctr = 14 . 19 ± 0 . 70 ; PTX3 = 19 . 09 ± 1 . 71 ; PTX3 + PD = 14 . 31 ± 0 . 70 , PD = 16 . 51 ± 1 . 17 . Number of neurons : Ctr = 14 , PTX3 = 14 , PTX3 + PD = 13 ; PD = 9 ; 3 independent experiments . Kruskall - Wallis test , p = 0 . 015 followed by Dunn " s test as indicated in figure , data are presented as a distribution plus mean ± SEM . Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov - Smirnov test : Ctr vs PTX3 : p < 0 . 0001 , D = 0 . 183 , PTX3 vs PTX3 + PD : p < 0 . 0001 , D = 0 . 251 , PTX3 vs PD : p < 0 . 0001 , D = 0G ) Representative images showing 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µmH ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [ ( GluA & Bsn ) / Bsn ] shows a statistically significant increase after PTX3 exposure , while pre - incubation with PD98059 prevents the PTX3 - dependent increase ( Ctr = 1 ± 0 . 042 , PTX3 = 1 . 499 ± 0 . 121 , PTX3 + PD = 1 . 093 ± 0 . 060 , PD = 1 . 017 ± 0 . 117 ; number of fields examined : 49 , 15 , 15 , 20 respectively ; one - way ANOVA analysis of variance , p = 0 . 0002 followed by post hoc Tukey test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± SEM ) I ) Quantification of surface synaptic GluA upon PTX3 application and simultaneous inhibition of β1 integrin and ERK1 / 2 signaling pathways reveals no additive effects with respect to single αCD29 or PD applications ( Ctr = 1 ± 0 . 061 ; PTX3 = 1 . 828 ± 0 . 151 ; αCD29 + PD + PTX3 = 0 . 766 ± 0 . 070 ; PTX3 + αCD29 = 1 . 043 ± 0 . 073 ; PTX3 + PD = 1 . 016 ± 0 . 061 ; PD = 0 . 969 ± 0 . 090 ; number of fields examined : 29 , 31 , 30 , 23 , 14 , 33 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Dunn ' s test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± SEM ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µmB) Blocking β1 integrins activity by using the specific anti-β1 Integrin monoclonal antibody prevents the PTX3-induced postsynaptic AMPARs recruitment (Ctr=1.000±0.074, PTX3=1.504±0.098, αCD29+PTX3=0.766±0.097, αCD29=0.810±0.108. Number of fields examined: 37, 33, 23, 24 respectively; one way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test; 3 independent experiments, data are presented as normalized mean values±SEM)C) Western blotting analysis of p-ERK levels on lysates from control and PTX3-treated neurons upon 30 minutes stimulation (Ctr=1±0; PTX3=1.453±0.195, 6 independent experiments, unpaired t-test, data are presented normalized on control and as mean±SEM)D) Representative mEPSC traces recorded from the indicated experimental conditions. E) mEPSC frequency quantitation showing that pre-incubation with PD98059 (30μM) completely prevents the PTX3-dependent increase of mEPSC frequency (Hz, Ctr=0.911±0.104; PTX3=1.748±0.273; PTX3+PD=0.518±0.055; PD=0.491±0.104. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. One-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. Data are presented as a distribution plus mean±SEM). F) inset: average mEPSC amplitude quantitation (pA), Ctr=14.19±0.70; PTX3=19.09±1.71; PTX3+PD=14.31±0.70, PD=16.51±1.17. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. Kruskall-Wallis test, p=0.015 followed by Dunn"s test as indicated in figure, data are presented as a distribution plus mean±SEM. Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov-Smirnov test: Ctr vs PTX3: p<0.0001, D=0.183, PTX3 vs PTX3+PD: p<0.0001, D=0.251, PTX3 vs PD: p<0.0001, D=0G) Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µmH) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [(GluA&Bsn)/Bsn] shows a statistically significant increase after PTX3 exposure, while pre-incubation with PD98059 prevents the PTX3-dependent increase (Ctr=1±0.042, PTX3=1.499±0.121, PTX3+PD=1.093±0.060, PD=1.017±0.117; number of fields examined: 49, 15, 15, 20 respectively; one-way ANOVA analysis of variance, p=0.0002 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM)I) Quantification of surface synaptic GluA upon PTX3 application and simultaneous inhibition of β1 integrin and ERK1/2 signaling pathways reveals no additive effects with respect to single αCD29 or PD applications (Ctr=1±0.061; PTX3=1.828±0.151; αCD29+PD+PTX3=0.766±0.070; PTX3+αCD29=1.043±0.073; PTX3+PD=1.016±0.061; PD=0.969±0.090; number of fields examined: 29, 31, 30, 23, 14, 33 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


Figure 5A ) PTX3 binds TSP1 and TSP - 2 but not TSP - 4 . Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP - 2 and TSP - 4 . Binding is reported as Absorbance at 450 nm ( Mean ± SD ) . Data are from one experiment out of three performedB ) N - terminal PTX3 binds TSP1 . Binding of PTX3 N - term domain was performed on TSP1 immobilized on plastic wells . Data are reported as Absorbance at 450 nm ( Mean ± SD ) and are representative of one out of two experiments performedD ) PTX3 binds the C - term proteolytic fragment of TSP1 . TSP1 and its fragments C - term , N - term , and Ca - 1 ( type III repeats ) were immobilized in plastic wells ( 5µg / ml ) and binding of PTX3 is reported as Absorbance at 450 nm ( Mean ± SD ) . Data refers to one out of two experiments performed with similar resultsE ) PTX3 and its N - terminal domain bind TSP1 C - terminal globular domain . 50 nM of P123 - 1 ( type I ' properdin ' repeats ) , E123 - 1 ( Type II EGF repeats ) , E123CaG - 1 ( type II repeats plus type III repeats and globular C terminus ) and TSP1 were immobilized in plastic well . Binding with PTX3 or N - terminal domain ( both at 220 nM ) was analyzed . Data are reported as Absorbance at 450 nm ( Mean ± SD ) and refers to one out of two experiments performed with similar resultsF ) Representative images of 14DIV control and PTX3 - treated cultures stained for the presynaptic marker bassoon ( blue ) and the microtubule protein tubulin ( red ) . Scale bar : 10μm . G ) Quantification of synaptic density ( Bsn / μm ) in the different experimental conditions ( Ctr = 0 . 385 ± 0 . 029 , PTX3 = 0 . 407 ± 0 . 032 , TSP1 = 0 . 721 ± 0 . 039 , PTX3 + TSP1 = 0 . 707 ± 0 . 037 , E123 = 0 . 764 ± 0 . 047 , PTX3 + E123 = 0 . 719 ± 0 . 041 . Number of fields examined : 86 , 80 , 75 , 72 , 46 , 55 respectively ; Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated in figure ; at least 3 independent experiments , data are presented as mean valuesH ) Representative images showing 14DIV neurons stained for surface AMPAR ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . I ) Quantification of the synaptic surface GluA density ( number of sGluA puncta / μm ) in the different tested conditions showing that GluA density increases in neuronal cultures treated with PTX3 or E123 + PTX3 but not in cultures treated with the full length TSP1 + PTX3 ( Ctr = 1 . 000 ± 0 . 055 ; PTX3 = 1 . 367 ± 0 . 074 ; TSP1 = 1 . 04 ± 0 . 061 ; PTX3 + TSP1 = 0 . 968 ± 0 . 076 ; E123 = 0 . 971 ± 0 . 063 ; PTX3 + E123 = 1 . 613 ± 0 . 077 . Number of dendrites examined : 103 , 80 , 88 , 57 , 51 , 62 respectively ; Kruskall - Wallis test , p < 0 . 0001 followed by Dunn ' s test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± SEM ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) PTX3 binds TSP1 and TSP-2 but not TSP-4. Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP-2 and TSP-4. Binding is reported as Absorbance at 450 nm (Mean±SD). Data are from one experiment out of three performedB) N-terminal PTX3 binds TSP1. Binding of PTX3 N-term domain was performed on TSP1 immobilized on plastic wells. Data are reported as Absorbance at 450 nm (Mean±SD) and are representative of one out of two experiments performedD) PTX3 binds the C-term proteolytic fragment of TSP1. TSP1 and its fragments C-term, N-term, and Ca-1 (type III repeats) were immobilized in plastic wells (5µg/ml) and binding of PTX3 is reported as Absorbance at 450 nm (Mean±SD). Data refers to one out of two experiments performed with similar resultsE) PTX3 and its N-terminal domain bind TSP1 C-terminal globular domain. 50 nM of P123-1 (type I 'properdin' repeats), E123-1 (Type II EGF repeats), E123CaG-1 (type II repeats plus type III repeats and globular C terminus) and TSP1 were immobilized in plastic well. Binding with PTX3 or N-terminal domain (both at 220 nM) was analyzed. Data are reported as Absorbance at 450 nm (Mean±SD) and refers to one out of two experiments performed with similar resultsF) Representative images of 14DIV control and PTX3-treated cultures stained for the presynaptic marker bassoon (blue) and the microtubule protein tubulin (red). Scale bar: 10μm. G) Quantification of synaptic density (Bsn/μm) in the different experimental conditions (Ctr=0.385±0.029, PTX3=0.407±0.032, TSP1=0.721±0.039, PTX3+TSP1=0.707±0.037, E123=0.764±0.047, PTX3+E123=0.719±0.041. Number of fields examined: 86, 80, 75, 72, 46, 55 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure; at least 3 independent experiments, data are presented as mean valuesH) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. I) Quantification of the synaptic surface GluA density (number of sGluA puncta/μm) in the different tested conditions showing that GluA density increases in neuronal cultures treated with PTX3 or E123+PTX3 but not in cultures treated with the full length TSP1+PTX3 (Ctr=1.000±0.055; PTX3=1.367±0.074; TSP1=1.04±0.061; PTX3+TSP1=0.968±0.076; E123=0.971±0.063; PTX3+E123=1.613±0.077. Number of dendrites examined: 103, 80, 88, 57, 51, 62 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


Figure 6A ) Representative images of 14DIV neurons stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . B ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a decrease of the surface synaptic AMPAR clusters upon chronic application of the PTX3 blocking antibody to mixed cultures ( Ctr = 1 . 000 ± 0 . 063 , PTX3 block = 0 . 694 ± 0 . 069 , isotype Ab = 0 . 876 ± 0 . 077 . Number of fields examined : 34 , 23 , 19 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure ; 3 independent experiments , data are presented as normalized mean values ± SEM ) C ) Representative images of 14DIV WT and PTX3 KO neurons stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) and tubulin ( red ) . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . D ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a reduction in PTX3 KO cultures with respect to WT ( WT = 1 ± 0 . 065 , KO = 0 . 771 ± 0 . 0 . 92 ; number of fields examined : 36 and 42 respectively ; Mann - Whitney test ; number of animals 5 WT and 6 PTX3 KO , data are presented as normalized mean values ± SEM ) E ) Examples of mEPSCs recorded in the indicated experimental conditions . F ) Quantitation of mEPSC inter - event interval showing a rescue of mEPSC frequency in PTX3 KO cultures treated with the N - terminal fragment of PTX3 ( WT = 1158 ± 34 . 67 ; WT + N - term = 979 . 1 ± 28 . 27 ; PTX3 KO = 1263 ± 38 . 99 ; PTX3 KO + Nterm = 1008 ± 30 . 14 . Number of neurons : WT , Ctr = 25 , WT + N - term = 29 ; PTX3 KO , Ctr = 24 ; PTX3 KO + N - term = 22 ; 3 independent experiments . Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated in figure , data are presented as mean ± SEM ) . G ) Quantitation of mEPSC amplitude showing a rescue in PTX3 KO cultures treated with the N - terminal fragment of PTX3 ( pA , WT = 20 . 85 ± 0 . 249 WT + N - term = 22 . 37 ± 0 . 234 ; PTX3 KO = 20 . 31 ± 0 . 264 ; PTX3 KO + Nterm = 23 . 8 ± 0 . 316 . Number of neurons : WT , Ctr = 25 , WT + N - term = 29 ; PTX3 KO , Ctr = 24 ; PTX3 KO + N - term = 22 ; 3 independent experiments . Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated in figure , data are presented asH ) Representative images of 14DIV PTX3 KO neurons ( Ctr , + N terminal PTX3 , + C terminal PTX3 ) stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) and tubulin ( red ) . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . I ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase in PTX3 KO treated with N terminal fragment of PTX3 but not with the C terminal fragment ( PTX3 KO = 1 . 000 ± 0 . 042 , PTX3 KO + C - term = 1 . 103 ± 0 . 070 , PTX3 KO + N - term = 1 . 428 ± 0 . 061 . Number of fields examined : 63 , 38 , 57 respectively ; Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test ; 3 independent experiments , data are presented as normalized mean valuesJ - L ( J ) Examples of mEPSCs recordings in WT and PTX3 KO ( littermates ) hippocampal slices at P8 - 9 showing that frequency ( K ) and amplitude ( L ) are significantly decreased in KO neurons with respect to WT ( Hz : WT = 1 . 54 ± 0 . 136 ; PTX3 KO = 0 . 912 ± 0 . 083 ; Mann Whitney test . pA : WT = 16 . 57 ± 1 . 334 ; PTX3 KO = 12 . 55 ± 1 . 037 ; unpaired t - test . Data are presented as a distribution plus mean ± SEM . WT : 18 cells , 4 mice ; PTX3 KO : 20 cells , 5 mice ) N ) Examples of mEPSCs recordings of P30 WT and PTX3 KO hippocampal slices . O ) Quantification of mEPSC frequency showing reduced frequency in PTX3 KO mice with respect to WT ( Hz : WT = 2 . 27 ± 0 . 115 ; PTX3 KO = 1 . 74 ± 0 . 136 . WT : 10 cells , 4 mice ; PTX3 KO : 11 cells , 5 mice . Unpaired t - test p = 0 . 008 . Data are presented as a distribution plus mean ± SEM ) . P , Q ( P ) No difference in the average amplitude is evident ( pA : WT = 11 . 95 ± 0 . 589 ; PTX3 KO = 10 . 69 ± 0 . 438 ; unpaired t - test p = 0 . 099 . Data are presented as a distribution plus mean ± SEM . WT : 10 cells , 4 mice ; PTX3 KO : 11 cells , 5 mice ) ; however the cumulative probability plot of amplitudes ( Q ) for mEPSCs in WT and PTX3 KO shows that there is a significant shift in the distribution by the Kolmogorov - Smirnov test ( p < 0 . 0001 , D = 0 . 112 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. B) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a decrease of the surface synaptic AMPAR clusters upon chronic application of the PTX3 blocking antibody to mixed cultures (Ctr=1.000±0.063, PTX3 block=0.694±0.069, isotype Ab=0.876±0.077. Number of fields examined: 34, 23, 19 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; 3 independent experiments, data are presented as normalized mean values ±SEM)C) Representative images of 14DIV WT and PTX3 KO neurons stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red). Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. D) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a reduction in PTX3 KO cultures with respect to WT (WT=1±0.065, KO=0.771±0.0.92; number of fields examined: 36 and 42 respectively; Mann-Whitney test; number of animals 5 WT and 6 PTX3 KO, data are presented as normalized mean values±SEM)E) Examples of mEPSCs recorded in the indicated experimental conditions. F) Quantitation of mEPSC inter-event interval showing a rescue of mEPSC frequency in PTX3 KO cultures treated with the N-terminal fragment of PTX3 (WT=1158±34.67; WT+N-term=979.1±28.27; PTX3 KO=1263±38.99; PTX3 KO+Nterm=1008±30.14. Number of neurons: WT, Ctr=25, WT+N-term= 29; PTX3 KO, Ctr= 24; PTX3 KO+N-term=22; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure, data are presented as mean±SEM). G) Quantitation of mEPSC amplitude showing a rescue in PTX3 KO cultures treated with the N-terminal fragment of PTX3 (pA, WT=20.85±0.249 WT+N-term=22.37±0.234; PTX3 KO=20.31±0.264; PTX3 KO+Nterm=23.8±0.316. Number of neurons: WT, Ctr=25, WT+N-term= 29; PTX3 KO, Ctr= 24; PTX3 KO+N-term=22; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure, data are presented asH) Representative images of 14DIV PTX3 KO neurons (Ctr, +N terminal PTX3, +C terminal PTX3) stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red). Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. I) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase in PTX3 KO treated with N terminal fragment of PTX3 but not with the C terminal fragment (PTX3 KO=1.000±0.042, PTX3 KO+C-term=1.103±0.070, PTX3 KO+N-term=1.428±0.061. Number of fields examined: 63, 38, 57 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn"s test; 3 independent experiments, data are presented as normalized mean valuesJ-L (J) Examples of mEPSCs recordings in WT and PTX3 KO (littermates) hippocampal slices at P8-9 showing that frequency (K) and amplitude (L) are significantly decreased in KO neurons with respect to WT (Hz: WT=1.54±0.136; PTX3 KO=0.912±0.083; Mann Whitney test. pA: WT=16.57±1.334; PTX3 KO=12.55±1.037; unpaired t-test. Data are presented as a distribution plus mean±SEM. WT: 18 cells, 4 mice; PTX3 KO: 20 cells, 5 mice)N) Examples of mEPSCs recordings of P30 WT and PTX3 KO hippocampal slices. O) Quantification of mEPSC frequency showing reduced frequency in PTX3 KO mice with respect to WT (Hz: WT=2.27±0.115; PTX3 KO=1.74±0.136. WT: 10 cells, 4 mice; PTX3 KO: 11 cells, 5 mice. Unpaired t-test p=0.008. Data are presented as a distribution plus mean±SEM). P, Q (P) No difference in the average amplitude is evident (pA: WT=11.95±0.589; PTX3 KO=10.69±0.438; unpaired t-test p=0.099. Data are presented as a distribution plus mean±SEM. WT: 10 cells, 4 mice; PTX3 KO: 11 cells, 5 mice); however the cumulative probability plot of amplitudes (Q) for mEPSCs in WT and PTX3 KO shows that there is a significant shift in the distribution by the Kolmogorov-Smirnov test (p <0.0001, D=0.112).


Figure 1 ( A ) The intracellular distribution of DOR is modified by starvation . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) , HBSS ( starvation ) , 50 μM chloroquine or 10 mM 3MA . The intracellular localization of DOR was analysed by immunofluorescence and is shown in red . The nuclei are shown in blue ( DAPI staining ) . Scale bars , 10 μm . ( B ) Endogenous DOR leaves the nucleus under starvation . C2C12 myoblasts were incubated for 30 min at 37 ° C with DMEM ( basal ) or HBSS ( starvation ) . DOR is shown in red and nuclei are in blue ( DAPI staining ) . Scale bars , 5 μm . ( C , D ) De ‐ activation of autophagy returns DOR to the nucleus . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) or HBSS ( starvation ) . Cells were then incubated with normal DMEM for an additional hour ( starvation + GM ) . The intracellular distribution of DOR was determined by immunofluorescence . DOR is shown in red and the nuclei are in blue . Scale bars , 5 μm . Cell lysates were also obtained and western blot assays were performed with specific antibodies . 3MA , 3 ‐ methyladenine ; DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; HBSS , Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm.(B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm.(C,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


Figure 2DOR localizes to autophagosomes when autophagy is activated . ( A ) Confocal images of HeLa cells transiently transfected with DOR and GFP ‐ LC3 , and incubated for 1h with DMEM , HBSS ( starvation ) , 2 μM rapamycin or HBSS containing 50 μM chloroquine . Nuclei were labelled with DAPI . Scale bars , 10 μm . ( B , C ) Confocal images of HeLa cells transiently transfected with DOR or GFP ‐ LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS . Scale bars , 10 μm . ( D ) Confocal images of HeLa cells transiently transfected with DOR and LAMP1 ‐ GFP , or with YFP ‐ LC3 and LAMP1 ‐ GFP . Scale bars , 10 μm . Contrast ‐ corrected merge RGB pictures and the Z ‐ projection of Product of the differences from the mean ( PDM ) images are shown in all panels . Colour scales of PDM images have different maximal values , so the different conditions are not comparable from these projections . Product of the differences from the mean ( PDM ) values closer to 1 show reliable colocalized pixels . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; GFP , green fluorescent protein ; HBSS , Hank ' s balanced salt solution ; LAMP1 , lysosomal ‐ associated membrane protein 1 ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm.(B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm.(D) Confocal images of HeLa cells transiently transfected with DOR and LAMP1‐GFP, or with YFP‐LC3 and LAMP1‐GFP. Scale bars, 10 μm. Contrast‐corrected merge RGB pictures and the Z‐projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal‐associated membrane protein 1; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


Figure 3 ( A ) DOR gain ‐ of ‐ function accelerates the degradation of proteins of middle - long half ‐ life . Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS . * Significant effects of starvation , P0 . 01 ; τsignificant effects caused by DOR overexpression , P0 . 01 . ( B ) DOR overexpression enhances autophagosomal formation . LC3 ‐ GFP ‐ positive vacuoles were counted in 100 transfected HeLa cells ( with GFP ‐ LC3 and TRα1 , or GFP ‐ LC3 and DOR ) . * Significant effects of starvation , P0 . 001 ; τsignificant effects of DOR overexpression , P0 . 001 . ( C , D ) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM ( basal ) or in HBSS ( starvation ) . Cells were processed by transmission electron microscopy . Arrows indicate autophagosomes . Insets are autophagosomes ( diameter , 407 ± 63 nm ) . Scale bars , 1 μm . Autophagosomes were counted in 50 randomly chosen transfected cells . * Significant effects of starvation , P0 . 05 ; τsignificant effects of DOR overexpression , P0 . 05 . DMEM ; Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; GFP , green fluorescent protein ; HBSS ; Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) DOR gain‐of‐function accelerates the degradation of proteins of middle-long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. *Significant effects of starvation, P0.01; τsignificant effects caused by DOR overexpression, P0.01.(B) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). *Significant effects of starvation, P0.001; τsignificant effects of DOR overexpression, P0.001.(C,D) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. *Significant effects of starvation, P0.05; τsignificant effects of DOR overexpression, P0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


Figure 4 ( A , B ) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA , DOR siRNA1 or DOR siRNA2 . Endogenous LC3 was detected by immunofluorescence . LC3 ‐ positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min . Scale bars , 10 μm . * Significant effects of starvation , P0 . 001 ; τsignificant effects caused by DOR knockdown , P0 . 001 . ( C , D ) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy . Arrows indicate autophagosomes . Insets are autophagosomes . Scale bars , 1 μm . Autophagosomes were counted in 50 randomly chosen cells . * Significant effects of DOR repression , P0 . 001 . ( E ) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA . * Significant effects of DOR knockdown , P0 . 01 ; τsignificant effects caused by 3MA , P0 . 01 . 3MA , 3 ‐ methyladenine ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A,B) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3‐positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. *Significant effects of starvation, P0.001; τsignificant effects caused by DOR knockdown, P0.001.(C,D) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. *Significant effects of DOR repression, P0.001.(E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. *Significant effects of DOR knockdown, P0.01; τsignificant effects caused by 3MA, P0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


Figure 5 ( A ) Decrease of mRNA levels in TubG4 , UAS ‐ CG11347 ‐ RNAi animals compared with TubG4 controls . All measurements were normalized to rp49 . * Significant change , P0 . 05 . ( B ) Lysotracker staining of fat bodies . No Lysotracker staining is observed in feeding early third ‐ instar larvae , whereas Lysotracker ‐ positive granules ( red ) accumulate in fat body cells of wandering late third ‐ instar controls ( upper panels , TubG4 / + ) . dDOR knockdown animals ( TubG4 , UAS ‐ CG11347 ‐ RNAi T4 , lower panels ) show decreased staining of Lysotracker ‐ positive granules in wandering late third ‐ instar larvae . Nuclei are shown in blue . Scale bars , 50 μm . ( C ) The number of Lysotracker ‐ positive puncta per unit area are shown for each genotype ( control and RNAi ) , normalized to the value for control cells processed in parallel . * Significant change , P0 . 05 . ( D ) Transmission electron microscopy images of fat body cells from wandering late third ‐ instar larvae . Developmental autophagy results in the accumulation of large autolysosomes ( AL ) in control ( TubG4 / + ) larvae , whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age . Control and RNAi fat bodies present lipid droplets ( LP ) with a ' striped ' appearance , which is an electron microscopy artefact . Scale bars , 5 nm . dDor , CG11347 , a homologue of DOR ; DOR , diabetes ‐ and obesity ‐ regulated gene ; mRNA , messenger RNA ; wt , wild type .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Decrease of mRNA levels in TubG4, UAS‐CG11347‐RNAi animals compared with TubG4 controls. All measurements were normalized to rp49. *Significant change, P0.05.(B) Lysotracker staining of fat bodies. No Lysotracker staining is observed in feeding early third‐instar larvae, whereas Lysotracker‐positive granules (red) accumulate in fat body cells of wandering late third‐instar controls (upper panels, TubG4/+). dDOR knockdown animals (TubG4, UAS‐CG11347‐RNAi T4, lower panels) show decreased staining of Lysotracker‐positive granules in wandering late third‐instar larvae. Nuclei are shown in blue. Scale bars, 50 μm. (C) The number of Lysotracker‐positive puncta per unit area are shown for each genotype (control and RNAi), normalized to the value for control cells processed in parallel. *Significant change, P0.05.(D) Transmission electron microscopy images of fat body cells from wandering late third‐instar larvae. Developmental autophagy results in the accumulation of large autolysosomes (AL) in control (TubG4/+) larvae, whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age. Control and RNAi fat bodies present lipid droplets (LP) with a 'striped' appearance, which is an electron microscopy artefact. Scale bars, 5 nm. dDor, CG11347, a homologue of DOR; DOR, diabetes‐ and obesity‐regulated gene; mRNA, messenger RNA; wt, wild type.


Figure 1 C ) . Western blot of LMF1 - partners after DSP crosslinking and affinity tag purification using LMF1 ' s C - terminal His tag . Tetracycline induces LMF1 expression . Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners . LMF1 complexes are labeled with asterisks and an arrow points to LMF1 D ) . Western blot of LMF1 - partners after pBPA photo - crosslinking and affinity tag purification . The DMSO panel does not include pBPA and serves as a negative control . Fractions were eluted with increasing concentrations of imidazole . An arrow points to LMF1 E ) . SYPRO orange protein stain of DSP crosslinked samples . Samples without tetracycline but with DSP and DTT ( negative control ) , and with tetracycline , DSP and DTT were sent for LC MS / MS . LMF1 complexes are labeled with asterisks and an arrow points to LMF1 F ) . SYPRO orange stained gel of pBPA samples . An arrow points to LMF1

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Figure 1 C). Western blot of LMF1-partners after DSP crosslinking and affinity tag purification using LMF1's C-terminal His tag. Tetracycline induces LMF1 expression. Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners. LMF1 complexes are labeled with asterisks and an arrow points to LMF1  D). Western blot of LMF1-partners after pBPA photo-crosslinking and affinity tag purification. The DMSO panel does not include pBPA and serves as a negative control. Fractions were eluted with increasing concentrations of imidazole. An arrow points to LMF1  E). SYPRO orange protein stain of DSP crosslinked samples. Samples without tetracycline but with DSP and DTT (negative control), and with tetracycline, DSP and DTT were sent for LC MS/MS. LMF1 complexes are labeled with asterisks and an arrow points to LMF1  F). SYPRO orange stained gel of pBPA samples. An arrow points to LMF1


Figure 2 A - B ) . HEK293 cells stably expressing LPL - V5 or PL - V5 were transfected with scrambled negative control siRNA ( NC ) or siRNA against LMF1 - interacting partners . Media fractions were probed for the lipase V5 tags , and lysate fractions were probed for V5 and the LMF1 interacting - partners . GAPDH is a loading controlsiRNA knockdowns were performed in triplicate , and lipase secretion was quantified relative to NC siRNA . Error bars indicate the standard error . Quantification of the percent of each protein remaining from three independent experiments . Error bars indicate the standard error . E ) HEK293 cells stably expressing LPL - V5 or PL - V5 were transfected with NC siRNA or siRNA against TRX . Lipase and TRX levels were tested as for Fig 1A F ) Txnip overexpression in HEK293 cells stably expressing LPL - V5 or PL - V5 hinders LPL , but not PL , secretion

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A-B). HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with scrambled negative control siRNA (NC) or siRNA against LMF1-interacting partners. Media fractions were probed for the lipase V5 tags, and lysate fractions were probed for V5 and the LMF1 interacting-partners. GAPDH is a loading controlsiRNA knockdowns were performed in triplicate, and lipase secretion was quantified relative to NC siRNA. Error bars indicate the standard error. Quantification of the percent of each protein remaining from three independent experiments. Error bars indicate the standard error. E) HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with NC siRNA or siRNA against TRX. Lipase and TRX levels were tested as for Fig 1A  F) Txnip overexpression in HEK293 cells stably expressing LPL-V5 or PL-V5 hinders LPL, but not PL, secretion


Figure 3 A ) . A Western blot against the C - terminal V5 tags of both lipases shows that LPL , but not PL , forms intermolecular disulfide bonds in cells . Lipase monomers are marked with a single arrow , and aggregates are marked with double and triple arrows . The LPL aggregates are not present when samples are treated with DTT . There is no PL in the pellet fraction because PL does not aggregate B ) . A Western blot of a non - reducing gel of the lysate and pellet fraction of LPL grown in cells lacking LMF1 ( HEK293 ∆ LMF1 ) , normal HEK293 cells or cells with additional LMF1 ( + LMF1 ) . LPL monomers and aggregates are marked as above C ) . Co - translational LPL folding was carried out in the presence of SP HEK293 or HEK293 ∆ LMF1 cells . At the indicated time points , AMS was added to reactions to differentiate reduced ( R ) and oxidized ( O ) LPL . The position of the stacker layer is indicated The amount of reduced / total LPL intensity was quantified . Each point is the average of three independent experiments , and error bars represent the standard error . Significance was determined by a 2 - tailed student ' s t - test . the amount of oxidized / total LPL ( E ) and the intensity of the stacker / total LPL ( F ) was quantified . Each point is the average of three independent experiments , and error bars represent the standard error . Significance was determined by a 2 - tailed student ' s t - test G and H ) . Data from ( D - F ) was combined to show the trends in LPL folding over time for reactions using HEK293 or HEK293 ∆ LMF1 cells . Each point is the average of three independent experiments , and error bars represent the standard deviation

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A). A Western blot against the C-terminal V5 tags of both lipases shows that LPL, but not PL, forms intermolecular disulfide bonds in cells. Lipase monomers are marked with a single arrow, and aggregates are marked with double and triple arrows. The LPL aggregates are not present when samples are treated with DTT. There is no PL in the pellet fraction because PL does not aggregate  B). A Western blot of a non-reducing gel of the lysate and pellet fraction of LPL grown in cells lacking LMF1 (HEK293∆LMF1), normal HEK293 cells or cells with additional LMF1 (+LMF1). LPL monomers and aggregates are marked as above  C). Co-translational LPL folding was carried out in the presence of SP HEK293 or HEK293∆LMF1 cells. At the indicated time points, AMS was added to reactions to differentiate reduced (R) and oxidized (O) LPL. The position of the stacker layer is indicated The amount of reduced/total LPL intensity was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test.the amount of oxidized/total LPL (E) and the intensity of the stacker/total LPL (F) was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test G and H). Data from (D-F) was combined to show the trends in LPL folding over time for reactions using HEK293 or HEK293∆LMF1 cells. Each point is the average of three independent experiments, and error bars represent the standard deviation