Fig. 5.

Induction of endothelial–mesenchymal transition (EndMT) by rapamycin treatment is caused by autophagy. a Schedule of stress-induced premature senescence (SIPS) induction, rapamycin treatment, and endothelial cell (EC) culture. To inhibit autophagy, human coronary artery endothelial cells (HCAECs) were pretreated with 100 nM bafilomycin A1 (BAF) for 1 h before rapamycin treatment. b Immunoblotting for the levels of LC3 was performed with cell lysates at day 0 in (a). β-ACTIN was used as a loading control. c Real-time PCR analysis of EndMT markers using cDNA derived from HCAECs at 72 h from (a). The results are shown after normalization to values obtained from control HCAECs (value = 1). Results are presented as means ± SD from three independent experiments. **p < 0.01. d Immunocytochemical staining performed in HCAECs at 72 h, as in (a). Representative images are shown (SM22, red; DAPI, blue). The histogram shows the mean ± SD percentage of SM22-positive cells. **p < 0.01. Control (Ctr): untreated cells