Figure 2
Disruption of IGF1R and ERBB2 signaling inhibits hESC self-renewal. (A) Treatment of H1 cells with the A12 anti-IGF1R blocking antibody led to decreased IGF1R expression on the cell surface as measured by flow cytometry. (B) Colony counting showed that the A12 antibody inhibited H1 cell proliferation (left). Arrows indicate that cultures were passaged on day 3 and 6. Cumulative colony counts increased in the presence of control antibody but not in the presence of A12. A12 also induced hESC differentiation (right) as measured by colony morphology (also see Figure S2). Error bars here and in panels C-H,J are standard deviation (SD). Flow cytometry for (C) SSEA-3 expression confirmed A12 treatment caused increased differentiation, but (D) not an increase in apoptosis. (E) Significant reduction of IGF1R mRNA in the GFP+ population of cultures transfected with an IGF1R-targeted shRNA, compared with either the GFP- population, or GFP+ cells transfected with a control shRNA. (F) Decline in the percentage of GFP+ cells in H1 cultures (left) transduced with a lentiviral vector containing a shRNA targeting IGF1R, rather than a control shRNA. Middle panel shows flow cytometry profiles that indicate stable expression of IGF1R and percentages of cells that are GFP+ in control shRNA–transduced cultures on days 1, 12, and 22, while cultures transduced with the IGF1R-targeted shRNA vector exhibited a decline in IGF1R expression and a reduction in GFP+ and GFP+/IGF1R+ cells. Right panel shows the average percentage of GFP+ cells at days 15, 19, and 22 was significantly lower in the IGF1R-targeted shRNA transduced culture. (G) A total of 50 μM AG825 inhibited proliferation of BG02 hESCs growing in CM. Triplicate cell counts from 2 independent experiments are shown. … and - - - indicate pretreatment cell counts from experiments 1 and 2, respectively. (H,I) Increasing concentrations of AG825 caused a dose-dependent reduction in the proportion of H1 hESCs in the G1 phase of the cell cycle, and (J) a moderate dose-dependent rise in apoptosis.

Disruption of IGF1R and ERBB2 signaling inhibits hESC self-renewal. (A) Treatment of H1 cells with the A12 anti-IGF1R blocking antibody led to decreased IGF1R expression on the cell surface as measured by flow cytometry. (B) Colony counting showed that the A12 antibody inhibited H1 cell proliferation (left). Arrows indicate that cultures were passaged on day 3 and 6. Cumulative colony counts increased in the presence of control antibody but not in the presence of A12. A12 also induced hESC differentiation (right) as measured by colony morphology (also see Figure S2). Error bars here and in panels C-H,J are standard deviation (SD). Flow cytometry for (C) SSEA-3 expression confirmed A12 treatment caused increased differentiation, but (D) not an increase in apoptosis. (E) Significant reduction of IGF1R mRNA in the GFP+ population of cultures transfected with an IGF1R-targeted shRNA, compared with either the GFP- population, or GFP+ cells transfected with a control shRNA. (F) Decline in the percentage of GFP+ cells in H1 cultures (left) transduced with a lentiviral vector containing a shRNA targeting IGF1R, rather than a control shRNA. Middle panel shows flow cytometry profiles that indicate stable expression of IGF1R and percentages of cells that are GFP+ in control shRNA–transduced cultures on days 1, 12, and 22, while cultures transduced with the IGF1R-targeted shRNA vector exhibited a decline in IGF1R expression and a reduction in GFP+ and GFP+/IGF1R+ cells. Right panel shows the average percentage of GFP+ cells at days 15, 19, and 22 was significantly lower in the IGF1R-targeted shRNA transduced culture. (G) A total of 50 μM AG825 inhibited proliferation of BG02 hESCs growing in CM. Triplicate cell counts from 2 independent experiments are shown. … and - - - indicate pretreatment cell counts from experiments 1 and 2, respectively. (H,I) Increasing concentrations of AG825 caused a dose-dependent reduction in the proportion of H1 hESCs in the G1 phase of the cell cycle, and (J) a moderate dose-dependent rise in apoptosis.

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