EMBO/SourceData huggingface dataset:
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 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 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.
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.
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
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
Figure 4Differential sensitivity of HEK293 cells containing and lacking LMF1 (A) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.Differential sensitivity of cld/cld and wt/cld MEFs (B) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test. C). The redox sensor ERroGFP-S4 shows that HEK293∆LMF1 cells have a more oxidized ER than HEK293 and HEK293+LMF1 cells both untreated and when stressed by expression of LPL-mCherry. A two-tailed student's t-test was used to calculate the significance of the difference in the ratio of oxidized/reduced GFP fluorescence intensity between cell types. Each data point is one of three spots measured per cell, 17 cells were measured per condition. Significance was determined by a 2-tailed student's t-test
Figure 5). Secretion of LPL-V5 from HEK293∆LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations. Lipase levels in the lysate and pellet, and LMF1 levels in the pellet (α-HIS), are also probed. GAPDH serves as a loading control.
Figure 6α-His Western blot to detect the redox state of LMF1. In the first two lanes samples were untreated and resolved in reducing or non-reducing loading dye. The next 4 lanes were subject to the indicated treatments prior to cell lysis then treated with AMS prior to non-reducing SDS-PAGE.α-His Western blot to detect acid trapped partners of LMF1. Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control.Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control. Samples were probed with an antibody for TRX.
Figure 7Bottom, center and top slices from a z-stack of a cld/wt or cld/cld MEF cell. YPet-tagged fibronectin is in green and mCherry-Sec61β, an ER marker, is in red. Note fibronectin assembly into the extracellular matrix in cld/wt cells. cld/wt cells had significantly higher amounts of fibronectin outside of the ER than cld/cld cells by a two-tailed student's t-test.Bottom, center and top slices show that LDLR is retained in the ER in cells HEK293∆LMF1 cells. LDLR-GFP is in green and mCherry-Sec61β is in red. Note the clumping of LDLR in the ER in HEK293∆LMF1 cells. The ratio of LDLR within the ER/total LDLR was significantly higher for HEK293∆LMF1 cells than HEK293 or HEK293+LMF1 cells by a 2-tailed student's t-test.
Figure 1A. Lysates of HK2 cells cultured in FM or STV(2h) were immunoprecipitated with anti-OFD1 (left) and anti-ATG13 (right) antibodies or IgG and analyzed by WB; ACTIN was used as loading control. n=3 independent experiments. IP=immunoprecipitation, FM=full medium, STV=HBSS starvation.B. Airyscan confocal analysis of endogenous FIP200/γTUBULIN and ATG13/γTUBULIN colocalization with lentiviral delivered HA-OFD1 in HK2 cells. Cells were cultured in HBSS (90min). Green, FIP200 and ATG13; red, HA-OFD1; Grey, γTUBULIN; blue, Hoechst for nuclei. Insets show magnifications and single-color channels of selected areas. Scale bar=3,5μm. C. Quantification of HA-OFD1/ATG13 and HA-OFD1/FIP200 colocalization, expressed as % of total OFD1 fluorescence (mean ± SEM). n=3 independent experiments; n=53 cells for ATG13 and n=39 for FIP200 were analyzed.
Figure 2A. Top, WB of ULK1-complex components in wt and KO-OFD1 cells assessed in FM (-) or STV (2,4h). Bands at same exposure, noncontiguous on the same gel. Bottom, ACTIN-normalized protein levels are expressed as fold increase compared with wt cells, represented by the dashed line (mean ± SEM); n=5 independent experiments. FM=full medium, STV=HBSS starvation.B. Top, wild-type and KO-OFD1 cells were incubated with 50µg/ml CHX. Immunoblots were probed with indicated antibodies. Bottom, quantification of βTUBULIN and ACTIN-normalized protein levels versus untreated conditions (-), data are expressed as mean ± SEM; n=4 independent experiments. CHX=Cycloheximide, ns=not significative.C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.D. Western Blot of ULK1 complex components in KO-OFD1 cells transfected with empty vector (Empty) or 3xFLAG-OFD1 (OFD1). Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM. n=5 independent experiments.E. Western Blot of ULK1-complex components in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or empty vector (Empty) and treated (+) or not (-) with SAR405 (10μM, 6h). ACTIN is the loading control. On the right, ATG13 levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.F. Representative confocal images of co-staining of 3xFLAG-OFD1 with ATG13 in KO-OFD1 cells incubated in HBSS, treated (+) or not (-) with Baf-A1(100nM, 90min). Green, ATG13; red, 3xFLAG-OFD1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=3 independent experiments, n≥100 cells.
Figure 3A. Middle. Lysates from 3xFLAG-OFD1 overexpressing HEK293 cells were added to beads with immobilized GST-fused LC3B, LC3BF52A-V53AdG, dNLC3BdG and GST alone. Right. Lysates from 3xFLAG-OFD1(OFD1) and empty vector (Empty) overexpressing HEK293 cells were added to GST-GABARAP-L1 and GST. Left. WB of total lysates (input). All panels display WB for FLAG, ACTIN is the loading control.B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample.D. Lysates from 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1∆LIR(OFD1∆LIR) and empty vector (Empty) overexpressing cells were added to GST-GABARAP-L1, GST-LC3B and GST.E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.
Figure 4A. Representative confocal images of 3xFLAG-OFD1 (OFD1) and 3xFLAG-OFD1∆LIR (OFD1∆LIR) co-staining with ATG13 in KO-OFD1 cells (HBSS,90min). Green, ATG13; red, FLAG; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=4 independent experiments, n≥100 cells.B. Western blot of ATG13 in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) or empty vector (Empty). (Right) ACTIN-normalized ATG13 protein levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.C. Top. OFD1 fragments (a,b and c). Bottom left. Lysates from empty vector (Empty), 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1a (OFD1a), 3xFLAG-OFD1b (OFD1b), 3xFLAG-OFD1c (OFD1c) overexpressing HEK293 cells were added to GST-ATG13; lysates from 3xFLAG-OFD1(OFD1) overexpressing HEK293 cells were added to GST as control. Right. WB of total lysates (input). All panels display WB for FLAG, HSP90 is the loading control. LIR=LC3 interacting region.D. Scanning confocal microscopy analysis of wt and KO-OFD1 cells incubated in HBSS (8h), treated (+) or not (-) with Baf-A1(50nM, 8h). Green, LAMP1; red, ATG13; blue, Hoechst for nuclei. Scale bar=6μm. Insets show higher magnification and single colour channels of the boxed area. (Bottom) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1/cell (mean ± SEM). n=30 WT cells, n=28 KO-OFD1 cells counted; three independent experiments.E. Time course of OFD1 levels in HBSS-starved wt HK2 cells treated with Baf-A1 (100nM) and collected at different time points. LC3B is the control for Baf-A1 treatment. (Right) Graphs show ACTIN-normalized OFD1 levels versus FM condition (-) expressed as mean ± SEM; n=4 independent experiments. STV=starvation, Baf-A1=bafilomycin, Ctrl=control.F. Western blot of FLAG in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) and treated or not (-) with Baf-A1 (100nM,2h). (Right) Quantification of GAPDH-normalized FLAG levels versus untreated (-) is expressed as mean ± SEM; n=3 independent experiments. Baf-A1=bafilomycin.