Figure 3. ITPR1 knockdown increases C2C12 cell proliferation through activation of the EGFR-Ras-ERK pathway. (A) ITPR1 knockdown and control C2C12 myoblasts were seeded at a density of 1x102 cells per well in 6-well plates. Cells were allowed to grow for 10 days until small colonies were clearly formed. Colonies were stained with crystal violet solution for 1 h. Cell-bound dye was eluted with a 1:1 solution of 0.1 M sodium citrate, pH 4.2, and ethanol, and absorbance of eluates determined at 590 nm (bottom left). Cell number was measured by direct counting of viable cells in a hemocytometer (bottom right). (B) ITPR1 knockdown and control C2C12 myoblasts were starved with or without mimosine for 24 h and released for 6 h before collection. Cells were permeabilized and stained with PI, prior to FACS analysis. The percentage of cells within each cell cycle phase (G1, S, and G2/M) was determined based on the DNA content. (C) Cell lysates from ITPR1 knockdown C2C12 myoblasts were analyzed by immunoblotting with anti-phospho EGFR, anti-Ras (GTP-bound form), anti-phospho ERK1/2, anti-ERK1/2, and anti- phospho CDK2 antibodies, with GAPDH as the loading control. s counted based on DAPI staining.