FIGURE 1.

Effect of UPS inhibition in 8220 CAPN3-deficient human myotubes. (A) Representative bright-field images of human control (NS-shRNA) and CAPN3 knockdown (shCAPN3) myotubes after 5 days in differentiation. Treatment with 5 nM BTZ was performed for 24 h where indicated. Scale bar: 50 μm. (B) Western blot analysis of Ca²⁺-handling proteins in control and CAPN3-deficient cells treated or not with BTZ. White line depicts non-continuous lanes within the same blot. Protein signals are normalized to MyHC and expressed as fold change over each control (NS-shRNA), which is represented as a discontinuous line in the bar chart. Data are expressed as mean fold-change ± SEM of n = 3 independent experiments; **p < 0.01 vs. non-treated shCAPN3 (ratio paired t-test). (C) Analysis of mRNA expression in CAPN3-deficient myotubes with or without BTZ treatment. Data expressed as mean fold-change ± SEM over NS-shRNA. n = 3 independent experiments (one-way ANOVA post hoc Tukey’s multiple comparisons test). (D) Representative pseudocolored images of human myotubes (NS-shRNA, shCAPN3, and shCAPN3 treated with BTZ) loaded with Fura-2AM. Scale bar: 25 μm. (E) Bar chart shows the resting intracellular calcium levels of human myotubes. Data expressed as mean % over NS-shRNA ± SEM based on calcium concentration levels. Dots represent individual experiments (n = 5) with a total of 120–160 myotubes analyzed per group (one-way ANOVA post hoc Dunnett’s multiple comparisons test).