FIGURE 5.

STIM2 is up-regulated in myotubes and participates to the refilling of Ca²⁺ stores. A, myoblasts were incubated with siSTIM2 for 5 h and immediately triggered to differentiate. 60 h afterward, multinucleated myotubes were formed, and cytoplasmic Ca²⁺ was assessed using Fura-2. Images illustrate Fura-2 measurements in multinucleated cells (nuclei are circled by discontinuous lines) at time “a” (resting Ca²⁺) and time “b” (max SOCE) of Fura-2 traces. One representative experiment (n = 3). B, Western blots illustrating the increase of STIM2 expression at various times of differentiation. Quantification was carried out of three Western blots performed on three different clones in proliferation and after 60 h of differentiation. α-Tubulin was used as loading control. C and D, 60 h after siSTIM2 transfection and differentiation induction (large multinucleated myotubes were present), cytoplasmic Ca²⁺ responses generated by successive KCl (65 mm) applications were assessed using Fura-2. siAllstar siRNA was used as control. E, peak Ca²⁺ responses to successive KCl pulses in siSTIM2-transfected myotubes. Ca²⁺ responses were normalized to the first response. F, Fura-2 measurements of remaining Ca²⁺ store content after seven pulses of 65 mm KCl. Ca²⁺ store content was assessed by application of 1 μm ionomycin in 250 nm external Ca²⁺. Results were normalized to those obtained in control conditions (three independent experiments).