Figure 5.

IRS-1 protein levels are decreased via mTOR signaling during insulin-supplemented hNSC culture. (a) Gradual reduction of IRS-1 protein levels over the culture period with insulin is prominent in hNSC cultures supplemented at the higher insulin concentration (left), but not in rat NSC cultures (right). hNSCs and rat NSCs were cultured in media supplemented with 0.22 and 4.3 μM of insulin as described in Figure 4. Protein samples were prepared 30 min after media change (adding freshly prepared insulin) at the time point indicated, and immunoblot analyses for IRS-1 were carried out. (b) Effect of the mTOR inhibitor rapamycin on apoptotic cell death of hNSCs at high insulin concentration. hNSCs (derived from H9 hESCs) were cultured for 4 days in the presence of 0.22 and 4.3 μM insulin, and 4.3 μM insulin+rapamycin (Rapa or R, 100 nM). Immunofluorescent staining was carried out for cleaved caspase-3 and the percentage of immunoreactive cells out of total DAPI+ cells was counted. (c) Treatment of rapamycin blocks the decrease of IRS-1 protein levels in hNSCs cultured with high concentration of insulin. *Significant difference from the high insulin sample (P<0.001, n=3 coverslips per group). Scale bars=40 μm