Fig. 3: mTOR Inhibition for Mature hiPSC-Epicardial Monolayers.

a Workflow to monitor Torin1 effects on hiPSC-derived epicardial monolayers. Created in BioRender. Lucena-Cacace, A. (2025) https://BioRender.com/qbjorue. b Western blot of Torin1-treated hiPSC-epicardial cells assesses mTOR target inhibition and quiescence markers (pAKT, pS6K, p53, p16, and p21; β-actin normalization). c Phase contrast microscopy shows day 31 Torin1-treated hiPSC-derived epicardium retains cobblestone morphology (scale bar: 200 µm). d Growth curve of Torin1-treated epicardium for 7 days (n = 3 biological replicates). e Immunocytochemical analysis of Torin1-treated hiPSC-epicardial cells for WT1, TBX18, and ZO-1 expression (scale bar: 200 µm). f, g Immunocytochemical analysis of Torin1-treated hiPSC-epicardial cells for MSLN and ZO-1 expression (scale bar: 200 µm), and immunofluorescence intensity quantification (n = 3 biological replicates). h MKI67 expression (left) and quantification (right) via Immunocytochemistry (scale bar: 200 µm; n = 3 biological replicates). i Immunocytochemistry for phosphor-histone 3 (pH3) expression (left) and quantification (right) (scale bar: 200 µm; n = 6 biological replicates). j Invasion assay on Torin1-treated hiPSC-epicardial cells. Representative images show invaded cells stained by crystal violet. The total number of celling invading through membrane are quantified. (scale bar: 500 µm; n = 3 biological replicates). k FACS analysis based on Pyrorin Y and Hoechst 33342 staining discriminates G0 cells. l Quantification and statistical analysis of quiescent hiPSC-epicardial cells after Torin1 treatment (Torin1, purple bars; n = 3 biological replicates). Data are presented as means ± s.e.m.; two-way ANOVA with Sidak’s multiple comparison test (d); two-tailed unpaired Student’s t-test (g–j, l). *p < 0.05, **p < 0.01, ***p < 0.001. Source data is provided as a Source Data file.