Figure 2. Effect of HL156A on IGF-I-mediated FoxO1 signaling in SAMP1/kl-/- MEFs. (A) Effect of SAMP1/kl-/- depletion on cell proliferation. SAMP1/kl+/+ and SAMP1/kl-/- MEFs were plated in 48-well plates at a density of 5 x 104 cells/well, and cell proliferation was evaluated using MTT assays after 24 and 48 h. (B) Comparison of IGF-1, FOXO1, and FOXO3 expression in SAMP1/kl+/+ and SAMP1/kl-/- MEFs. Total RNA was extracted from SAMP1/kl+/+ and SAMP1/kl-/- MEFs. cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). (C) The mRNA levels of IFGR and FOXO1 were determined in SAMP1/kl+/+ and SAMP1/kl-/- MEFs. Total protein was extracted, and the protein levels of IFGR, p-IGFR, and FOXO1 were measured by Western blot. Actin was used as a loading control. (D) The effect of HL156A on IGF-1 signaling. SAMP1/kl+/+ and SAMP1/kl-/- MEFs were incubated in the absence or presence of HL156A (10 or 20 μM) for 24 h, and total RNA was then isolated and subjected to RT-PCR analysis to determine the mRNA levels of IGF-I, FOXO1, and mTOR. (E) Changes in the mRNA abundance of FOXO1 were analyzed with real-time RT-PCR analysis in HL156A-treated SAMP1/kl+/+ and SAMP1/kl-/- MEFs. (F) HL156A induced FOXO1 expression. Cells were treated with HL156A for 24 h, and FOXO1 levels were then determined using Western blot analysis. (G) The effect of HL156A on IGF-I-mediated Akt/mTOR/p70S6K signaling. The expression of IGF-I-mediated Akt/mTOR/p70S6K proteins in SAMP1/kl+/+ and SAMP1/kl-/- MEFs treated with HL156A (10 or 20 μM) for 24 h. (H) Immunoblotting analysis of GSK3β, Akt, mTOR, and p70S6K expression in SAMP1/kl-/- MEFs treated for 24 or 48 h with 20 μM HL156A. (I) Effects of HL156A on MAPK activation in SAMP1/kl-/- MEFs.