Figure 5. Inhibition of IGF-1 expression with PPARγ inverse agonists blocks MEC cell growth and induces apoptosis.

(A and B) PPARγ-response element (PPRE)-driven (A, left) and IGF-1 promoter-driven (A, right) luciferase reporter assay showing repression of basal transcriptional activity of endogenously expressed PPARγ in HEK293-CMV^TetRTetO^C1/M2 cells expressing C1/M2. Representative dose-response curves show cells treated with increasing concentrations of the PPARγ inverse agonist SR10221 for 24 h before measuring luciferase activity. (B) Dose-response curves showing viability of three C1/M2-positive cell lines (HMC1, HMC3A, and HMC3B) treated with increasing concentrations of PPARγ inverse agonists (SR10221, SR2595, and T0070907). IC₅₀ values were calculated using a non-linear curve fit (log[agonist] versus response, four parameter-variable slope) in GraphPad Prism. The experiment was performed in biologic triplicate (n = 3 technical replicates for each experiment), with one representative experiment shown.

(C) Quantitative real-time PCR analysis of IGF-1, PGK1, and PKM2 expression in HMC3A cells treated with vehicle (DMSO) or SR2595. Relative fold expression is shown normalized to RPL23 mRNA levels. Data are presented as the mean ± SEM (n = 3 biologic replicates, 3 technical replicates each; Student’s t test; ***p ≤ 0.001).

(D) Left, western blot showing expression of IGF-1 protein in HMC3A cells treated with increasing concentrations of SR2595. The blot is representative of three independent biologic replicates. Right, quantification of IGF-1 protein levels. Band intensities were normalized to β-actin and are shown as the fold change in band intensity relative to the vehicle-treated condition. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; ns, not significant, *p ≤ 0.05).

(E) Cell proliferation assay showing relative confluency of HMC3A cells treated with DMSO or SR10221. 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 ≤ 0.001). Data were collected using the IncuCyte live-cell imager; see STAR methods for details.

(F) Colony formation in HMC1 cells treated with DMSO or SR10221 at 1/2 IC₅₀ concentration or IC₅₀ concentration. A colony is defined as a cluster of ≥50 cells. Data are presented as the mean ± SEM (n = 3 biologic replicates with 3 technical replicates each; Student’s t test; ***p ≤ 0.001).

(G) Tumorsphere formation in HMC3A cells treated with DMSO or 1/2 IC₅₀ SR10221. 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 ≤ 0.01, ***p ≤ 0.001).

(H) Apoptosis levels (measured as the percentage of cleaved caspase-3/7) in HMC3A cells treated with DMSO or SR10221 at IC₅₀ concentration or 23 IC₅₀ concentration. 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 ≤ 0.001, ****p ≤ 0.0001).

See Table 1 for a summary of the IC₅₀ values. See also Figure S6.