EMBO/SourceData huggingface dataset:
Figure 4Quantification of the relative frequency of myofibre cross-sectional areas (CSAs) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti-IGF2R for 4 and 9 weeks (a).(b) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti-IGF2R administration. The quantification of regenerating and M-cadherin-positive satellite cells per section is shown. One-way ANOVA test, **p<0.01; ***p<0.001; ****p<0.0001.(c) Representative WB analysis of neonatal myosin heavy chain (neonatal MHC) expression and quantification expressed as the ratio of neonatal MHC/β-actin in untreated mdx mice and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; ***p<0.0001 (n=10 mice per group).(d) Specific force (maximal tetanic force normalized to cross-sectional area, Po/CSA) from TA and VM muscles in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).SERCA activity quantified as ATPase hydrolysis activity vs. min (e) in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).
Figure 5(a) Representative WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β-actin expression in the TA and VM muscles in untreated C57BL mice, untreated mdx mice and mdx treated with low and high doses of anti-IGF2R for 4 and 9 weeks. Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A/β-actin in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001 (n=10 mice per group).(b) Representative WB analysis of CAMKII, p-CAMKII and GAPDH expression quantified and shown as CAMKII/GAPDH and p-CAMKII/CAMKII ratios in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01 (n=10 mice per group).(c) Representative WB analysis of PKCα, p-PKCα and β-actin expression levels quantified and expressed as the PKCα/β-actin and p-PKCα/PKCα ratios in arbitrary units (n=10 mice per group). All values are expressed as the mean±SEM.
Figure 6WB analysis performed in TA and VM muscles of mdx mice treated with anti-IGF2R using antibodies against CD20, pSer+pThr and vinculin. Densitometry analysis of data expressed as the pSer+pThr/CD20 and CD20/vinculin ratio in arbitrary units. One-way ANOVA *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group)(a).Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti-IGF2R using anti-IGF1R, anti-pospho-IGF1R and GAPDH. Densitometry analysis of data expressed as the pIGF1R/IGF1R ratio normalized to GAPDH content in arbitrary units. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group) (b). All values are expressed as the mean±SEM.
Figure 7Corrosion casting results showing the linearization of capillaries in IGF2R-treated VM mdx muscles. Pericytes around capillaries are shown in the insert box (a). Scale bars=100μm.Representative immunofluorescence results (b) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown.Representative immunofluorescence quantification (c) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown. Scale bars=75μm. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean± SEM.(d) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti-IGF2R. Densitometry analysis of data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean ± SEM.(e) Representative Ve-cadherin (red) and CD31 (green) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti-IGF2R for 4 weeks. Scale bars=25 μm.
Figure 1C, Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.
Figure 3Hypocotyl lengths in SD condition of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week.C-E, Average CCA1:LUC bioluminescence traces of indicated plant lines. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. White and gray regions indicate subjective light and dark period. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in period between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test).
Figure 4A-C, PIL1, AT5G02580 and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data is representative of 3 biological trials.
Figure 6B, Measurement of TOC1-GFP protein turn-over in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Data information: TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ.C, Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with student's t test, different letters indicate significant differences (p<0.05). Data from 3 independent trials were averaged.
Figure 7B, Quantification of interactions between TOC1/5X-TAP and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TOC1/5X were immunoprecipitated with IgG resins followed by HRV-3C protease digestion. TOC1/5X and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated TOC1/5X to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, Student's t test).C, ChIP-qPCR assay of native promoter driven TOC1-GFP and 5X-GFP's residence at target promoters in toc1 vs. nf-yc3/4/9 toc1 (c349t1) mutants. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant differences (* p<0.05, *** p<0.0001, Student's t test), n.s= not significant.
Figure 8A, Co-IP assay of HDA15-TAP with TOC1-GFP and 5X-GFP in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and TOC1 were detected by α-MYC and α-GFP, respectively. B, Quantification of the interaction strength in A. Results from 5 independent trials were averaged. Error bar indicates SEM, asterisk indicates significant difference (*p<0.05, student's t test). C and D, Hypocotyl lengths and free-running periods of indicated plants lines. Data of ~75 plants from 3 independent experiments were averaged, different letters indicate statistically significant differences (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test). The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value.E, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 independent experiments were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
Figure 9A and B, ChIP-qPCR assay of HDA15's (A) and NF-YC9's (B) residence at target promoters in presence or absence of TOC1 and PRR5. GFP-HDA15 and GFP-NF-YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col-0, toc1 single and prr5 toc1 double mutant grown in SD condition. Results were normalized by % input of PIL1 coding region (PIL1 CDS). Data from at least 3 independent experiments were averaged, error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.001, *** p<0.0001, Student's t test).C and D, Dual luciferase assay of the repression activity of GFP-TOC1 and GFP-5X on promoter of AT5G02580 (C) and CDF5 (D) in protoplasts of toc1 and hda15toc1 (h15t1) mutants. AT5G02580:LUC, CDF5:LUC reporters and the effectors (empty GFP, GFP-TOC1, and GFP-5X) were co-transfected to toc1 and hda15 toc1 protoplasts with 35S:Renilla-LUC as internal control. Data are means ± SEM (n = 5 biological trials) relative to empty GFP which is set as 1. Different letters indicate statistically significant differences (student's t test, p<0.05).
Figure 10A, Quantification of interactions between TAP-PRR5 and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TAP-PRR5 was immunoprecipitated with IgG resins followed by HRV-3C protease digestion. PRR5 and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated PRR5 to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, student's t test).C, ChIP-qPCR assay of native promoter TOC1-GFP and 5X-GFP binding to the target promoters at ZT14 in toc1 vs. prr5 toc1 (p5t1) mutant. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicate significant differences (Student's t test; * p<0.05), n.s= not significant.D, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 biological trials were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
Figure 1A. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/µg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time-points post-infection (n=4 per time-point). nd: not detected. Horizontal lines indicate the median.B. Follow-up of the clinical signs of mice under different treatments. Heatmaps were established based on a progressive 0-7 clinical score scale (0: no apparent changes | 1: ruffled fur | 2: slow movement, hind limb ataxia | 3: apathy | 4: monoplegia | 5: hind limb paralysis, tremors | 6: paralysis, conjunctivitis/ keratitis, urine staining of the haircoat of the perineum | 7: death). Each line represents one animal throughout time.C. Body weight progression of mice under different treatments. Mice were weighted on a daily basis during the treatment administration, and then twice a week up to 100 days post-infection. All the mice were equipped with iPrecio pumps weighting ca. 3.3 g. Each line represents one animal throughout time.D-E. Mouse behavioral testing. The tested animals were non-infected (n=3 mice with iPrecio pump + n=4 age-related mice without iPrecio pump), infected non-treated (n=5), infected and treated at 7 dpi (n=5, mice #9 to #13), and infected and treated at 8 dpi (n=5, mice #24 to #28). (D) Open field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase) and after the remission of clinical signs for the survivors (20, 30 and 60 dpi). Horizontal lines indicate the median. Kruskal-Wallis followed by the Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann-Whitney test (20, 30 and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman r = -0.768) with the clinical score at 8 dpi. *P<0.05, **P<0.01, ***P<0.001. Treatments description= NI: non-infected (n=5); NT: infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).
Figure 2A. Cumulative Kaplan-Meier survival curves of mice treated at different time-points post-infection. Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. **P<0.01, ***P<0.001. Treatments description= infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).B. Cumulative Kaplan-Meier survival curves of all the treated animals that presented with clinical signs (n=31, all treatments combined). Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. ****P<0.0001.C. Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups.D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors.Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: , (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.
Figure 3A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C(N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images. Data information: N-R: Iba1 IHC to detect microglial cells O-P are high magnifications of N.
Figure 2 A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test  C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;
Figure 3 A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test. *P ≤ 0.05, **P ≤ 0.01
Figure 5Electron microscope images of mitochondria from SH-SY5Y e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experimentsElectron microscope images of mitochondria fro MEF WT, MEF PINK1 K as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experimentsElectron microscope images of mitochondria fro Hela an e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experimentsElectron microscope images of mitochondria fro Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments For flies, more than 200 mitochondria from each group were counted from 5 different experimentsRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments
f1(a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification. Left panel: HeLa cells growing in control (C: nutrient-rich media) or starved in HBSS (H) for 4 h were lysed and subjected to IP with ROCK1 cross-linked beads. Proteins bound were eluted and detected by SDS-PAGE, followed by silver staining that presents precipitated proteins identified by LC-MS/MS. Right panel: HeLa cells grown in control media or HBSS (4 h) were collected, whole cell lysates prepared and immunoprecipitated with ROCK1 cross-linked beads. The resulting immune complexes were divided into two, and were subjected to SDS-PAGE and analysed for Beclin1 (MES buffer) and ROCK1 (MOPS buffer). Inputs and IP were analysed by western blotting. HBSS-treated cell extracts were used for IgG IP controls. Whole cell lysates (10%) were run for inputs. Total cell lysates were rerun and analysed for LC3 and β-actin.(b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression.(c) Interaction of YFP-ROCK1 and CFP-Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells. Graph represents FRETAverage ratio changes (YFP-ROCK1/CFP-Beclin1) upon starvation. Data represents mean±s.e.m.; n=6. Three different experiments were performed. For the control, the right top graph represents CFP intensity over time (corrected for photobleach), and the right bottom right represents YFP intensity (corrected for photobleach). In both control FRET experiments, no significant change was observed in fluorescence, in the presence or absence of HBSS medium. P values were calculated using Student's t-test.