Figure 3. Inhibition of IGF-1R blocks MEC cell growth and induces apoptosis.

(A) Left, western blot showing expression of total and phosphorylated (Tyr1135) IGF-1R protein in HMC1 cells treated with BMS-754807 or PPP. The blot is representative of three independent biologic replicates. Right, quantification of phosphorylated (Tyr1135) IGF-1R protein levels. Band intensities were normalized to β-actin and to total IGF-1R and are shown as the fold change in phosphorylated band intensity relative to the untreated condition. Data are presented as the mean ± SEM (n = 3 biologic replicates; one-way ANOVA with Holm-Sidak post hoc test; ns, not significant, *p ≤0.05).

(B) Cell proliferation assay showing relative confluency of HMC3A cells treated with DMSO or PPP. GC₅₀ values for each condition are shown in hours and indicate the time required for cells to reach 50% confluency. ND, not determined. The experiment was performed in biologic triplicate (n = 4 technical replicates per experiment) with one representative experiment shown (mean ± SEM; Benjamini-Hochberg pairwise comparisons;**p ≤0.01, ***p ≤0.001). Data were collected using the InCucyte live-cell imager; see STAR methods for details.

(C) Number of colonies formed by HMC3A cells treated with DMSO or PPP. A colony is defined as a cluster of ≥50 cells. Representative images of wells treated with DMSO and PPP (IC₅₀ concentration) are shown to the left. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; ****p ≤0.0001). See Table 1 for a summary of the IC₅₀ values.

(D) Representative images of HMC3A tumorspheres on day 1 (top) and 7 days after treatment with DMSO (vehicle control) or increasing concentrations of PPP.

(E) Tumorsphere formation in HMC3A cells treated with DMSO or increasing concentrations of PPP. The percent change in the tumorsphere area is calculated as [TumorArea_DayX]/[TumorArea_Day1] × 100. >50 individual tumorspheres per condition were tracked for each experiment. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; ****p ≤0.0001).

(F) Apoptosis levels (measured as the percentage of cleaved caspase-3/7) in HMC3A cells treated with DMSO or PPP. Data were collected via flow cytometry and normalized to a non-stained control for each condition. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; **p ≤ 0.01).

See also Figure S4.