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. 2021 Oct 14;16(11):2752–2767. doi: 10.1016/j.stemcr.2021.09.009

Figure 4.

Figure 4

Biochemical characterization of autophagic flux in FKRP mutant myotubes

(A) Western blot for LC3B-II and LAMP1 in FKRP KO myotubes and WT control. Cells were assessed under basal or HBSS, with or without 100 μM chloroquine (CQ). ACTA1 and α-tubulin were used as differentiation and loading controls, respectively.

(B) Left panel shows quantification for LC3B-II, of western blots shown in (A) normalized to α-tubulin. Right panel shows autophagic flux calculated as the difference in LC3B-II levels of HBSS-treated myotubes with and without CQ. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by RM two-way ANOVA followed by Sidak's multiple comparison test (left) and paired Student's t test (right). Error bars are mean ± SEM for four independent experiments.

(C) Respective quantification for LAMP1 of western blots shown in (A) normalized to α-tubulin. ∗p < 0.05 by RM two-way ANOVA followed by Sidak's multiple comparison test. Error bars are mean ± SEM for four independent experiments.

(D) Representative western blot for LC3B-II and LAMP1 in WT and WWS myotubes. Cells were assessed under basal or starving conditions (HBSS), with or without 100 μM CQ. ACTA1 and α-tubulin were used as differentiation and loading controls, respectively.

(E and F) Quantitative analysis of (D) for LC3B-II (E) and LAMP1 (F) normalized to α-tubulin. Right panel in (E) shows flux calculated as the difference in LC3B-II levels of HBSS-treated myotubes with and without CQ. ∗p < 0.05 by RM two-way ANOVA followed by Sidak's multiple comparison test and paired Student's t test in (E) right panel. Error bars are mean ± SEM for four independent experiments.

(G) Western blot for LC3B-II and LAMP1 in WWS and WWS corrected myotubes. Cells were assessed under basal or starving conditions (HBSS), with or without 100 μM CQ. ACTA1 and α-tubulin were used as differentiation and loading controls, respectively.

(H and I) Graph shows quantification of LC3B-II (G) and LAMP1 (I) normalized to α-tubulin. Right panel in (E) shows flux calculated as the difference in LC3B-II levels of HBSS-treated myotubes with and without CQ. ∗p < 0.05, ∗∗p < 0.01 by RM two-way ANOVA followed by Sidak's multiple comparison test and paired Student's t test in (E) right panel. Error bars are mean ± SEM for four and six independent experiments.