Figure 1

BVRA depletion markedly increases the ROS generation in young HDF cells. (A) Young and senescent HDF cells were treated with 1 mM H₂O₂ for the indicated periods. After lysis, the cell extract was prepared, and the reductase activity was measured at pH 8.7. The activity analysis was repeated using three separate preparations of HDF ^*P ≤ 0.05 and ^**P ≤ 0.01 when compared with young HDFs. (B) Young HDFs were transfected with three different siRNAs of BLVRA and control siRNA using Oligofectamine. After 72 h, cells were harvested. Protein expression of BLVRA were analyzed by Western blotting using anti-BLVRA antibody (upper panel). All proteins were normalized to the densitometric signal of the actinin loading control (lower panel). (C) After transfection with BLVRA siRNA or a control siRNA for 72 h, cells were stained with dichlorofluorescein diacetate (DCF-DA), fixed and immediately analyzed by FACS. The data are mean ± SEM of three independent experiments. A double asterisk (^**) denotes P < 0.01 in Student's t-test. (^†) denotes P < 0.01 compared with non-transfected cells (ANOVA, Dunnett was used as post-test). (D) Effect of BLVRA deficiency on intracellular ROS level in young and senescent cells undergoing oxidative stress. After transfection with BLVRA siRNA or a control siRNA for 72 h, cells were treated with 1 mM H₂O₂ with/without NAC (20 mM), then stained with dichlorofluorescein diacetate (DCF-DA), fixed and immediately analyzed by FACS. The data are mean ± SEM of three independent experiments. A double asterisk (^**) denotes P < 0.01 in Student's t-test. DCF: dichlorofluorescein.