EMBO/SourceData huggingface dataset:
Figure 5(D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.
Figure 6(B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01). (C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001).
Figure 7(M) Statistical results of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in (D-F). More than twenty-five midguts were examined for each group. Error bars indicate SEM. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).
Figure 1(A) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm.(C) Representative images showing the myogenic differentiation of MuSCs isolated from 1.5 month old mdx mice, as assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with mdx FAPs, which were pretreated with DMSO or GW4869 (GW 10mM added to FAPs 30 minutes before starting the co-culture with MuSCs). Scale bar = 50 μm. (D) Graph showing the fusion index of MuSCs in the conditions described in (C), (n=5, biological replicates). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), ** p < 0.01; Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-cultured with FAPs not treated (DMSO), # p<0.05; § represents statistical analysis by 2Way Anova test; §§§ p < 0,001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.(E) Representative images of PKH-67 (green) and DAPI (blue) staining in a transwell co-culture between MuSCs and FAPs isolated from 1.5 month old mdx mice. FAPs exposed to PKH-67 prior to the co-culture with MuSCs. Scale bar = 50 μm. (F) Graph showing the percentage of PKH-67 positive MuSCs after the co-culture with FAPs, (n=3, biological replicates). Star (*) indicates statistical analysis by t-test;**** p < 0.0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.(G) Representative images of myogenic differentiation of mdx MuSCs, as assessed by immunostaining for MyHC (green). MuSCs were cultured either alone (-) or incubated with EVs isolated from 1.5 month old mdx mice (EVs FAPs) treated with vehicle (CTR) or exposed to TSA in vivo (TSA) or in vitro (TSA vitro). Scale bar = 50 μm. (H) Graph showing the fusion index of MuSCs in the condition described in (G), (n=3, biological replicates). Star (*) indicates statistical analysis by Tukey test on the % of n>5 (% of nuclei that were MyHC+ inside myotubes containing more than 5 nuclei) relative to MuSCs cultured alone (-), **p <0.01, ****p<0,0001; Hash (#) indicates statistical analysis by Tukey test relative to EVs FAPs CTR, ##p <0.01, ###p<0,001; ns= not significant; § represents statistical analysis by 2way Anova test, §§§§p < 0,0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.
Figure 2(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01.(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.(E) Representative images of miR-206-3p (violet) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages: 1 year (n=2), 4 years (n=3) and 9 years old (n=2). (F) Graph representing miR-206-3p violet area quantification relative to (E). (G) Representative images of immunofluorescence for eMyHC (red) and Laminin (green) in conditions described in (E). (H) Graph showing the percentage of eMyHC positive fibers of condition described in (G). (I) Representative images of Masson's Trichrome staining in conditions described in (E). (J) Graph showing the fibrotic area quantification of conditions described in (I).
Figure 3(A) Representative images of Sca-1 (green), Laminin (white), DAPI (blue) immunofluorescence, and miR-206-3p (violet) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior (TA) of young mdx mice (CTR) and after TSA treatment (TSA) (0.6 mg/kg/day for 15 days by i.p.) (n=6). Scale bar = 25 μm.(C) Graph representing the miR-206-3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice (CTR), mdx young and old mice (control -CTR- and TSA treated) (n=4). Star (*) indicates Tukey analysis compared to wild type mice (CTR), ****p < 0.0001; hash (#) indicates Tukey analysis compared to mdx young CTR, #### p<0.0001. § represents statistical analysis by Anova test. §§§§ p<0,0001.(D) Representative images of CD90 (green), Dapi (blue) and miR-206-3p (violet) immunohistochemistry in brachial biceps bioptic samples of DMD patient before (CTR) and after Givinostat treatment (GIV). Scale bar = 50 μm.(F) Graph showing the relative expression of miR-206-3p in EVs isolated from a human population of muscle resident cells enriched in FAPs (ehFAPs) isolated from biopsies of two DMD patients (DMD-1 and DMD-2) before (CTR) and after (GIV) Givinostat treatment, Two technical replicates are shown.
Figure 5A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01.(D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01.(E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesicles(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm.
Fig 1A. In response to a 1 s light pulse, cac alleles lack on transients at day 3 and day 33.B. Quantification of depolarization amplitudes (top) and on amplitudes (bottom). Data are presented as means ± standard error of the mean (SEM).
Fig 2A. TEM sections of 3- and 27-days-old photoreceptor terminals of flies. Terminals of 27-days-old flies show terminal expansion, loss of capitate projections (CPs) and active zones (AZs) and increased number of mitochondria per terminal. Scale bars, 500 nm.B. Inset from A representing the photoreceptor terminal boxed in white. Yellow arrow indicates double-membraned autophagosome, red indicates amphisome, and blue indicates two fusion-primed AVs. Scale bars, 500 nm.C-E. Quantification of CPs, AZs, and mitochondria per cartridge, respectively. Data are presented as means ± SEM.F. Quantification of the different AVs per cartridge. The mutant terminals accumulate late stage AVs and fusion-primed AVs (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). Data are presented as means ± SEMG. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left sideH. The level of p62 is increased in cacJ brain. I. Quantification of H. Data are presented as means ± SEM. (*: p < 0.05).
Fig 3Autophagy defects observed in cac mutants are not due to defects in neurotransmitter release, but due to defects in lysosomal fusion.TEM sections of 1-day-old and 30-days-old photoreceptor terminals of flies raised in 12 h light/dark conditions. A, B, D, and E. stj and Vha100-1 (V0) mutant terminals accumulate AVs upon aging. G, H. n-Syb mutant photoreceptor terminals show increased SVs, but not a significant accumulation of AVs and fusion-primed AVs. J, K, M and N. Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation. C, F, I, L, O. Quantification of the different AVs for each genotype (**: p < 0.01). Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as "n = " in the figure. Data are presented as means ± standard deviation (SD). Scale bars, 1 μm.
Fig 4A. Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg-la mice have progressive PC and granule cell loss upon aging. Scale bar, 200 μm.. B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of micecerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-lamicecerebellum at day 25 (B) and the Cacna2d2du-2Jmice at day 50 (D). Scale bar, 20 μmC, E. The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg-la and Cacna2d2du-2J mice are swollen and accumulated with numerous mitochondria, autophagosome/MVBs-like structures (red arrows), and various cytoplasmic vesicles or aberrant membrane structures, including stacks of cisternal membranes (blue arrows), and folded ER (yellow arrows). Scale bars for right two panels are 2 μm. Other scale bars are 500 nmF, G. LC3II and p62 are slightly increased in 15-days-old Cacna1atg-la mice and 50-days-old Cacna2d2du-2J mice. b, d are quantification of a and c. Data are presented as means ± SEM. (*: p < 0.05).
Fig 5A. CACNA1A co-localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.C. Quantification of LAMP1 positive organelles that are also CACNA1A positive. Data are presented as means ± SD. (ns: not significant).D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.
Fig 6A. LysoTracker Red staining of the primary cerebella neurons from Cacna1atg-la mice and WT controls. The control cells show big bright punctae, while the mutant cells have much dimmer and smaller punctae. B, C and D. Quantification of LysoTracker relative intensity ("n" represents the counted cell number), LysoTracker positive punctae number and size per cell ("n" represents the counted image number). E. Lysosomal marker LAMP1 co-localizes with autophagosome marker LC3 in CTL but not Cacna1atg-la mutant cells. Scale bar, 20 μm. F. Quantification of LC3 and LAMP1 co-localization. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001).
Fig 7A. DQ-BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells. Scale bar, 20 μm. B. Quantification of DQ-BSA and LAMP1 co-localization in CTL and Cacna1atg-la cells. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001) C. Bepridil but not ω-agatoxin TK (ω-Aga TK) treatment reduces lysosomal fusion in cultured cerebellar cells. DQ-BSA (Green) labeled late endosomes co-localize with LAMP1 (Red) labeled lysosomes with/without ω-Aga TK (1 µM) or Bepridil (10 µM) treatment. Scale bar, 20 μm. D. Quantification of DQ-BSA and LAMP1 co-localization with ω-Aga TK or Bepridil treatment. Data are presented as means ± SD. (ns: not significant; **: p < 0.001).
Figure 1Representative current traces from oocytes expressing wild-type (WT) and double cysteine mutant NMDARs before and after DTE treatment. For each mutant, traces are normalized (in height, not in current amplitude) to the maximal response obtained after DTE treatment on wild-type (WT) receptorsSummary of the DTE-induced potentiation of WT and double cysteine mutantsImmunoblots from Xenopus oocytes expressing either wt or mutant subunits. M1 indicates the GluN1 monomer (~110 kDa); M2 the GluN2B monomer (~180 kDa); D1/1 the GluN1 homodimer (~220 kDa); and D1/2 the GluN1/GluN2B heterodimer (~290 kDa). Lower panels: immunoblots in reducing conditions (+ β-mercaptoethanol). N.I., non-injected oocytes
Figure 2Assessment of receptor channel activity using MK-801 inhibition kinetics. Representative current traces from oocytes expressing either wild-type (WT) or GluN1-E698C/GluN2B-L795C mutant receptors in response to 10 nM MK-801 during agonist application. Responses were scaled to the current amplitude obtained before MK-801 application. Inset, mono-exponential fits of MK-801 wash-in and wash-out. Note the strikingly faster kinetics in mutant receptors, both at the onset and offset of MK-801Relative MK-801 inhibition on- and off-rate constants (kon and koff). All values were normalized to the value obtained for WT GluN1/GluN2B receptors
Figure 3Zinc, ifenprodil and pH dose-response curves of wild-type (WT) GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors. For comparison, zinc and ifenprodil dose-response curves of GluN1/GluN2B receptors lacking the whole GluN2B NTD (GluN1 WT/GluN2B-delNTD) are also shown (dashed lines; data from Rachline et al, 2005)Spermine (200 µM, pH 6.3) potentiation of WT GluN1/GluN2B and mutant GluN1-E698C/GluN-L795C receptors. Spermine (200 µM, pH 6.5) sensitivity is also shown for WT GluN1/GluN2B receptors and receptors lacking either the GluN1 (GluN1-delNTD/GluN2B WT) or GluN2B (GluN1 WT/GluN2B-delNTD) NTD (data from Mony et al, 2011)Glutamate and glycine dose-response curves of WT GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors
Figure 5MK-801 inhibition kinetics. On-rate constants (kon) of inhibition by 10 nM MK-801 on wild-type (WT) and mutant receptors. All values are normalized to that obtained for WT GluN1/GluN2B receptors
Figure 6Representative recordings of patches containing one single wild-type (WT) or GluN1-E698C/GluN2B-L795C receptor. The bottom trace is an expanded view. C, closed channel; O, open channelSingle channel properties (equilibrium channel open probability, mean open time, mean closed time) of the WT and super-active GluN1-E698C/GluN2B-L795C receptorsClosed (left) and open (right) time histograms for WT (upper panels) or super-active GluN1-E698C/GluN2B-L795C (lower panels) receptors. Closed time histograms were best fit with 5 exponentials, whereas open time histograms were best fit with 2 exponentials (see Online Methods and Appendix Fig S8). Smooth lines are associated exponential fits
Figure 1(A-C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP-LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP-LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation).(D and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015).(F) Comparison of isogenic β‐cateninWT/− and β‐cateninHCT116 cells. Upper panel: western blotting showed decreased LC3‐II in HCT116β‐catenin cells. Lower panel: western blotting confirmed by quantification using densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent treatments, *P=0.046).Source data for this figure is available on the online supplementary information page.
Figure 2(A) Protein expression of p62 increased following β‐catenin knockdown under normal and starved conditions in HT29 cells.(B) Comparison of HCT116 expression in isogenic HCT116β‐cateninWT/− and HCT116β‐catenin−/ΔS45 cells by western blotting showed decreased p62 expression in β‐catenin−/ΔS45 cells.(C) LC3 puncta number per cell during starvation and nutrient addback post starvation. Left panel: LC3 puncta number reduces in non‐targeting (NT) siRNA control with nutrient addback. Right panel: LC3 puncta number reduces in β‐catenin knockdown cells with nutrient addback (mean±s.e.m. of three independent treatments; n>200 cells in >20 fields of view per experiment). Western blotting for β‐catenin confirmed knockdown.(D) Western blotting of LC3 and p62 in HT29 cells following β‐catenin knockdown and starvation with lysosomal inhibitors. LC3‐II and p62 protein expression showed an increase with β‐catenin siRNA in the presence of autophagy flux inhibitors chloroquine (10 μM) or bafilomycin A1 (100 nM; applied for the final 30 min of the 8 h) compared to β‐catenin knockdown alone. Quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of four independent experiments). See also Supplementary Figure S1A.Source data for this figure is available on the online supplementary information page.
Figure 3(A-D) LC3 staining (red) in mouse intestinal crypt and villus epithelium increased (white arrowheads) 2 days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (C and D) compared to control β‐catenin+/lox‐villin‐creERT2 mice (A and B). Further magnification of the areas marked by white squares is shown (lower panels).(E-H)p62 staining (green) increased in the crypt and villus epithelium2 days after β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (G and H) compared to control in β‐catenin+/lox‐villin‐creERT2 mice (E and F). DAPI staining (blue) identifies nuclei. β‐Catenin deletion was confirmed by immunofluorescence ( Supplementary Figure S1B). Red and green channel levels were adjusted post acquisition for clarity (equal level changes applied across the entire figure). Further magnification of the areas marked by white squares is shown (lower panels).(I and J) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 and LC3‐II protein expression 2 (I) and 4 (J) days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice compared to control β‐catenin+/lox‐villin‐creERT2 mice.Source data for this figure is available on the online supplementary information page.
Figure 4(A) Relative p62/SQSTM1 mRNA levels by quantitative real‐time‐PCR (qRT-PCR) increased following β‐catenin knockdown in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐Catenin knockdown was confirmed by western blotting.(B) Relative p62 mRNA levels by qRT-PCR decreased with β‐cateninS33Yoverexpression after 24 h of starvation in HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐CateninS33Y overexpression was confirmed by western blotting.(C) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C.(D) Overexpression of Fzd6 (green, left panel) increased p62 (red, right panel) protein expression in HEK293T cells. As an internal control, cells not overexpressing Fzd6 are shown in the field of view.(E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling.(F) Relative p62 mRNA levels (48 h) by qRT-PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).(G) Upper panel: Wnt3a (200 ng/ml) treatment of HCT116 β‐cateninWT/− cells (24 h) decreased LC3‐II and p62 protein expression by western blotting. Lower panel: quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent experiments).Source data for this figure is available on the online supplementary information page.