Fig. 2.

Insulin suppresses Rax expression on SFEBq culture. (A–C) FACS analysis of Rax–GFP knocked-in ES cells that were cultured by SFEBq for 7 days in gfCDM (A), GMEM/KSR (B), and gfCDM + 7 μg/ml insulin C. (D–F) Annexin V⁺ apoptotic cells were analyzed (day 4) with (E) or without (D) insulin. For positive control, cells were preincubated with 10 μM etoposide for 4 h (F). (G) Time-window analysis of insulin's effect on Rax–GFP⁺ percentages by FACS on day 7. Insulin (7 μg/ml) was removed (upper graph) or added (lower graph) as shown in the left bars. The ratio of the Rax–GFP⁺ percentages in the presence of insulin to that of SFEBq/gfCDM culture (row 1; referred to as 1.0) is shown in the graphs at the right. (H–J) Effects of inhibitors on SFEBq-cultured ES cells in gfCDM with or without insulin (7 μg/ml) were analyzed by FACS on day 7. LY294002 (H and I), Akt inhibitor VIII (H and J), PD98059 (10 μM), or DMSO (vehicle) was added on day 2. Statistical significance vs. the control (gfCDM + insulin with DMSO) was evaluated by the Dunnette test. *, P < 0.05; **, P < 0.01. (K–R) qPCR analysis of insulin's effect on day 7 was performed as in Fig. 1 P–X.