Extended Data Figure 5: Forced expression of FOXO1 does not induce apoptosis, senescence, autophagy or energy distress in cultured ECs.
From: FOXO1 couples metabolic activity and growth state in the vascular endothelium
a, Immunoblot analysis and quantification of FOXO1 protein levels in AdCTL and AdFOXO1CA–Flag transduced HUVECs (n = 20). b, ATP levels in ECs 24 h after transduction with AdCTL or AdFOXO1CA (n = 7). c, Western blot images and quantification of AdCTL- or AdFOXO1CA–Flag-transduced HUVECs showing that FOXO1CA does not alter the phosphorylation of AMPKα (Thr 172) or of its substrate ACC (Ser 79). Oligomycin (Oligo), positive control. TUB, tubulin. n = 10. d, Western blotting of AdCTL- or AdFOXO1CA–Flag-transduced HUVECs illustrating that overexpression of FOXO1CA does not induce apoptotic cell death. Cleaved caspase3 (CASP3) and PARP served as markers of apoptosis. Cycloheximide (CHX) and TNF-α (TNF) costimulation, positive control. e, Analysis of senescence-associated genes by microarray demonstrating that senescence markers were not significantly changed or even downregulated in FOXO1CA-overexpressing ECs. n = 3. f, Images of β-galactosidase stainings in AdCTL- and AdFOXO1CA-transduced HUVECs showing no increase in senescence-associated β-galactosidase activity (SABG). g, Densitometric quantification of the LC3-II to LC3-I ratio in AdCTL- or AdFOXO1CA-transduced HUVECs (n = 10). h, Immunofluorescence analysis of AdCTL- and AdFOXO1CA-transduced HUVECs (both coexpressing GFP) using LC3 and GFP antibodies. Chloroquine (CQ), positive control. DAPI, endothelial nuclei. Data in a–c, e and g represent mean ± s.d. Two-tailed unpaired t-test. *P < 0.05; ****P < 0.0001; NS, not significant.
