Fig. 7
VPA rescues differentiation arrest and ?1-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 ?m. 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 ?ídák'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 ?ídák'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 ?m. 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 ?1-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 ?1-integrin trafficking. h Top: expression of ?1-integrin in WT and Mtm1-KO C2C12 cells at the indicated differentiation time points (n?=?3 independent experiments). Bottom: quantification of ?1-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 ?1-integrin of WT, Mtm1-KO, Mtm1-KO VPA-treated C2C12 cells after 10 days of differentiation (n?=?3 independent experiments). Bottom: quantification of ?1-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 ?1-integrin distribution from 10-days differentiated WT (k), Mtm1-KO (l) and Mtm1-KO C2C12 myotubes?±?250 µM VPA (m). Scale bar, 10 ?m. Bottom: higher magnifications of the boxed area shown on the top. Scale bar, 5 ?m. n Quantification of ?1-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 ?ídák's multiple comparisons test |