FIGURE 2.

Uncoupled SOCE activation in C2C12 cells after silencing of JP1 and JP2. (A) Representative traces of percentage changes in ratio of fluorescence at excitation of 360 and 380 show that addition of 20 μM TG (arrow) induced passive Ca²⁺ release from SR Ca²⁺ stores (left panel). In JP shRNA-treated C2C12 myotubes (gray, n = 4), SR Ca²⁺ stores are significantly less than that in myotubes transfected with pU6-EGFP-control plasmids (black, n = 5) indicated by reduced TG-releasable Ca²⁺ (right panel). (B) Quenching of intracellular Fura-2 fluorescence by the entry of extracellular Mn²⁺ (0.5 mM) reveals graded and sigmoidal activation of SOCE in C2C12 myotubes transfected with control plasmids (black, n = 5). In JP shRNA-treated C2C12 myotubes (gray) the sigmoidal phase of Fura-2 quenching by Mn²⁺ was completely absent, indicating defective SOCE (n = 4). (C) To examine the changes in the maximum degree of SOCE activation, C2C12 cells were incubated with TG for an extended period of time to ensure complete depletion of their SR Ca²⁺ stores. Compared with the control (n = 4, maximum quenching rate is −9.96 ± 0.88%/min), the maximum activation of SOCE was reduced in siRNA-transfected C2C12 myotubes (n = 5, maximum quenching rate is −4.99 ± 1.40%/min) by ∼50%. p < 0.005.