ELECTRICAL STIMULATION PREVENTS DELETERIOUS EFFECTS OF CHEMOTHERAPY ON CULTURED MYOTUBES-SUPPLEMENTAL DATA

SUPPLEMENTAL FIGURE LEGENDS:Supplemental Figure 1. (A) Raw tracings of Fluo-2 AM-derived intracellular Ca2+ cycling (top portion of panel) and contractions (bottom portion of panel) of myotube during 20V, 12 ms STIM at 1Hz and its response to tetrodotoxin (TTX; 10µM; red dotted line), showing cessation of all Ca2+ cycling and contraction. Note that random spikes of contractile activity of the myotube upon application and following TTX are due to debris floating through the field where cellular landmarks were chosen to track contractile activity. These are most prevalent upon initial application of TTX due to fluid shear effects. (B) Raw tracings of Fluo-2 AM-derived intracellular Ca2+ cycling (top portion of panel) and contractions (bottom portion of panel) of myotube during 20V, 12 ms STIM at 1Hz and its response to N-benzyl-p-toluene sulphonamide (BTS; 50µM; 1st red dotted line), followed by tetrodotoxin (TTX; 10µM; 2nd dotted red line). This tracing shows that BTS causes cessation of contraction without altering Ca2+ cycling, with the latter being abolished by TTX.Supplemental Figure 2. Effects of 1 d of doxorubicin (DOX; 0.2 µM; gray bars) or vehicle (DMSO; open bars) administration, with or without electrical field stimulation (STIM; 20V, 12 ms pulses at 1Hz for 1 h/d) on Akt phosphorylation. STIM reduced phosphorylation of Akt (p-Akt; T308) after 1 d (PSupplemental Figure 3. Effects of doxorubicin (DOX; 0.2 µM) administration, with or without capsaicin administration (CAP; 100 nM), on mitochondrial ROS production and content. A) CAP administration was unable to mitigate the effects of 3 d of DOX administration to increase ROS production (n=6/bar; PSupplemental Figure 4. Effects of 1 hour of doxorubicin (DOX; 0.2 µM; gray bars) or vehicle (DMSO; open bars) administration, with or without electrical field stimulation (STIM; 20V, 12 ms pulses at 1Hz for 1 h/d) on AMPK phosphorylation. (A) No effect of DOX or STIM for 1 h was found on phosphorylation of AMPK (p-AMPK; T172). Representative gel images of p-AMPK and total AMPK expression are shown at the top of the panel for a sub-set of replicates. All data are mean ± SEM, with individual data points shown with each bar. SUPPLEMENTAL VIDEO LEGENDSSupplemental Video 1. Pseudocolor video of intracellular Ca2+ cycling in d7 myotube measured by Fluo-2 AM in response to 20V, 12 ms pulses at 1Hz. Time signatures for each image are provided at the bottom (frame rate=40/s, scale bar=10 µm).Supplemental Video 2. Contractile dynamics in d7 C2C12 myotubes with 2 μm latex beads (1:1000) in response to 20V, 12 ms pulses at 1Hz. Time signatures provided at the bottom (frame rate=10/s, scale bar=10 µm).