Fig. 5: Rapamycin decreases the expression of the mTOR target proteins p-S6K1 and p-eIF4E, in FAP1 organoids.

A Confocal immunofluorescence images of day 48 FAP1 colon organoids for p-S6K1 and p-eIF4E. This experiment was repeated twice, and 2-3 organoids were analyzed from each experiment. Representative images are presented. Scale bars- 50 µm. B Western blot of phosphorylated S6K1 and phosphorylated eIF4E for FAP1 and FAP2 organoids on day 48, in the presence/absence of rapamycin. The experiment was repeated twice, quantified by ImageJ, and normalized to the non-phosphorylated protein. ****P < 0.0001, t test. C Immunohistochemical analysis of p-S6K1 and p-eIF4E in day 48 colon organoids derived from a control hESC line (H9), and FAP1 with/without rapamycin. Normal colon tissue served as a positive control. Scale bar- 100 µm. Two independent biological experiments were performed, and representative data are shown. D Confocal immunofluorescence analysis of FAP1 cells differentiated in monolayer from day 20 to day 30, with and without rapamycin. Three independent biological experiments were performed. Scale bars- 50 µm.