Figure 3.

Cell cycle distribution of RAX knockdown αT3 cells. (A) αT3 cells expressing an shRNA construct targeting RAX (1199) or a non-targeting control (NT) were plated following 72 h of puromycin selection and counted at 24-h time points. (B) Proliferation of αT3 and LβT2 cells stably expressing one of two RAX shRNAs (1199 or 1459) or a non-targeting control was determined as in (A); data were then fit to the equation Y = Y₀e^KT, where T is hours after plating, Y is cell number (or absorbance) and Y₀ is starting cell number (or absorbance), to determine the proliferation rate K. Results are shown as mean ± SEM, αT3 NT (n=7), 1199 (n=5), 1459 (n=5), LβT2 NT (n=4), 1199 (n=4), 1459 (n=3); * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.0001. See Table 2 for statistical analysis. (C) αT3 cells expressing one of two RAX shRNAs (1199 or 1459) or a non-targeting control were analyzed for RAX expression by western blot following 72 h of puromycin selection (T=0 h in time course shown in (A)). (D) Cells from the experiment shown in (E) were stained with trypan blue to determine viability. Results are shown as mean ± SEM (n=3). (E) αT3 cells expressing RAX (1199) shRNA or a non-targeting control were selected with puromycin and stained with PI at 24-h time points to measure DNA content and cell cycle distribution by flow cytometry. Percentage of cells in G₀/G₁, S and G₂/M phases are shown as mean ± SEM (n=3).