Gsmtx 4

MPCUCs were loaded with the fluorescent Ca²⁺ indicator Fura-2 AM (10 μM; Life Technologies, Carlsbad, CA) with 0.04% Pluronic F-127 (Sigma-Aldrich, St. Louis, MO) for 60 min at room temperature. The cells were then washed with balanced salt solution (BSS). 10 μM GsMTx4 (Peptide Institute, INC., Osaka, Japan) and 10 μM RR (WAKO) were applied directly into the chamber. Measurement of [Ca²⁺]_i was performed for 5 min at pre- and post-stretch stimulation, using previously described methods with slight modifications^{7 (link),8 (link)}. The ratio-changes were calculated by subtracting basal values from the peak values. The analysis conditions were required to satisfy the following three criteria. 1; The basal values of the Fura2 ratio must be less than 1.5. 2; The cell-cultures must be larger than the field view of the microscope. 3; When the cells were not fixed on the silicon chamber bottom at post-stretch, the cells around them were excluded from the analysis.

Constructs encoding murine nAChR subunits (α, β, γ and δ) were kindly provided by Dr Stanley C. Froehner (University of Washington, Seattle). All expression plasmids were verified by DNA sequencing. GsMTx-4 (toxin from the Chilean Rose Tarantula, Grammostola spatulata) was obtained commercially from Peptide Institute, Inc. (Osaka, Japan).

C2C12 myoblasts described previously (4 (link)) or mouse primary myoblasts were maintained and induced to differentiate into myotubes, and they were treated with STO-609 (Calbiochem), EGTA (Wako), GsMTx-4 (Peptide Institute), Yoda1, nifedipine (Wako), or tranilast (Tokyo Chemical Industry). Adenoviral vectors for LacZ and mouse KLF15 were described previously (35 (link)). Mouse KLF15 and negative control siRNAs were obtained from Invitrogen and were delivered into cells with the use of the Lipofectamine RNAiMAX transfection reagent (Invitrogen). Isolation of total RNA and quantitative RT-PCR analysis were performed as previously described (4 (link)). Data were normalized by the amount of 36B4 mRNA. The sequences of PCR primers are provided in Supplemental Tables 2 and 3. Immunoblot analysis was performed with antibodies against STAT3 (4904, Cell Signaling Technology) and against Tyr⁷⁰⁵-phosphorylated STAT3 (9131, Cell Signaling Technology). Uncropped immunoblots are presented in Supplemental Figure 12.