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Fig. 7
VPA rescues differentiation arrest and β1-integrin alterations in Mtm1-KO myotubes. a Brightfield of MHC and DAPI staining of differentiated C2C12 myotubes (D10) showing that VPA rescues the aberrant differentiation and reduced size observed in Mtm1-KO cells. Scale bars, 100 μm. a–c are higher magnifications of the indicated areas shown as boxes. b Average area of MHC labeled WT, Mtm1-KO and Mtm1-KO VPA-treated cells after 10 days of differentiation. Data are represented as Mean ± SEM, n = 5, 5 fields per independent experiment, each point represents one field of view, ****p < 0.0001 and **p < 0.01, one-way ANOVA test and Šídák's multiple comparisons test. c Relative directionality of WT, Mtm1-KO and Mtm1-KO VPA-treated cells after 10 days of differentiation. Data are represented as Mean ± SEM, n = 5 fields per independent experiment, each point represents one field of view, ns not significant and ****p < 0.0001, one-way ANOVA test and Šídák's multiple comparisons test. d BioID workflow for the identification of an MTM1 interactome. e MHC staining of WT, Mtm1-KO and Mtm1-KO expressing MTM1-miniTurbo-flag myotubes after the indicated days of differentiation, showing that miniTurbo does not affect MTM1 function. Scale bar, 100 μm. f Identification of candidate interactors by quantitative mass spectrometry. Volcano plot depicting quantified proteins (gene names). The x axis shows the average fold change (log2) in protein abundance in MTM1-miniTurbo samples in comparison to miniTurbo controls, and the y axis shows the –log10 (p value); data for both were determined by results from three independent experiments. Significantly enriched proteins in the MTM1-miniTurbo condition are displayed in orange, MTM1 bait protein displayed in blue, and known MTM1 partners in green. Proteins (gene names) related to β1-integrin trafficking in purple. g Enrichment (abundance ratios as log2 values (MTM1-miniTurbo—miniTurbo)) of peptides from three independent experiments (mean) related to MTM1 and β1-integrin trafficking. h Top: expression of β1-integrin in WT and Mtm1-KO C2C12 cells at the indicated differentiation time points (n = 3 independent experiments). Bottom: quantification of β1-integrin expression analyzed by Western. Data are represented as Mean ± SEM, n = 3, *p < 0.05 and **p < 0.01 according to Student’s t test. i Top: expression of β1-integrin of WT, Mtm1-KO, Mtm1-KO VPA-treated C2C12 cells after 10 days of differentiation (n = 3 independent experiments). Bottom: quantification of β1-integrin expression. ns not significant and **p < 0.01 according to Student’s t test. j Top: expression of Talin1 of WT, Mtm1-KO, Mtm1-KO VPA-treated C2C12 cells after 10 days of differentiation (n = 3 independent experiments). Bottom: quantification of Talin expression. ns not significant and **p < 0.01 according to Student’s t test. Top: representative confocal images of β1-integrin distribution from 10-days differentiated WT (k), Mtm1-KO (l) and Mtm1-KO C2C12 myotubes ± 250 µM VPA (m). Scale bar, 10 μm. Bottom: higher magnifications of the boxed area shown on the top. Scale bar, 5 μm. n Quantification of β1-integrin vesicles density (Mean ± SEM, n = 3, 5 fields per independent experiment, each point represents one field of view, ****p < 0.0001 according to one-way ANOVA test and Šídák's multiple comparisons test