ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties

Figure 4

ALDH2 silencing or inhibition induces the expression of senescence markers in HUVECs. (a, b) Images and area of cells in siCTR and siALDH2 ECs cultured in EBM-2 supplemented with 2% FBS for 48 h. Data are expressed as a square pixel of cells analyzed using ImageJ. Quantification of 70 cell areas for each condition is reported. vs. siCTR. Two-way ANOVA was used. (c) SA-β-Gal quantification, expressed as a fold increase in positive cells for SA-β-Gal activity ± SD vs. PD 5. vs. PD 5. (d, e, f) Western blot analysis of a pattern of senescent markers (d, left: p21 and p53 or right: Egr-1 and c-Myc) in HUVECs at PD 5 (#5, PD 5) and PD 21 (#21, PD 21) or (e, p21 or f c-Myc, Egr-1, and p53) in siCTR and siALDH2 HUVECs, 48 h posttransfection (#5, PD 5; #21, PD 21). Representative blots of 3 with similar results are shown (e, g, h). Quantification of immunoblot in (d), (e), and (f). Data are reported as an ADU fold increase vs. siCTR (e, h) or vs. PD 5 (g). (e) and vs. siCTR. (g) and vs. PD5. (h) , , and vs. siCTR. (i) Images of SA-β-Gal staining of siCTR and siALDH2 ECs and PD 21 groups obtained with a Leica DMI4000 microscope. Images of HUVECs at PD 21 were reported as a positive control. Scale bar: 250 μm. The insets show boxed areas in detail. (j) Cells were transfected with siRNA for 2 or 6 days. The transfection was repeated every 72 h. SA-β-Gal quantification, expressed as a fold increase ± SD vs. siCTR of positive cells for SA-β-Gal activity. and . (k) Western blot analysis of senescent markers in HUVECs at PD 5 in the presence/absence of daidzin (10 μM) for 48 h (#5, PD 5). Representative blots of 3 with similar results are shown. (l) SA-β-Gal quantification in ECs treated or not with daidzin (10 μM) for 48 h, expressed as a fold increase in positive cells for SA-β-Gal activity ± SD vs. untreated cells. vs. untreated cells. (m) Cells were transfected with siRNA for 2 or 6 days in the presence/absence of NAC (5 mM). The transfection was repeated every 72 h. Cells were pretreated for 30 min with NAC, before the treatment with 2% of FBS. The pretreatment with NAC and the treatment with 2% FBS were repeated every 3 days. SA-β-Gal quantification, expressed as a fold decrease ± SD vs. untreated cells positive for SA-β-Gal activity. . Images are representative of results obtained with siALDH2 B.

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