Research Paper Volume 13, Issue 14 pp 17978—18005

Figure 3. Knockdown of HDAC2 or HDAC7 induces senescence in AG04431 cells. Cells at early passage were transfected with control siRNA (non target, siNT), HDAC2 siRNA (siHDAC2) or HDAC7 siRNA (siHDAC7) during 24 hr and biomarkers of senescence were analysed every day during 7 days. (A) Representative Western blots showing HDAC2 and HDAC7 total protein abundance at different times (24-168 hr) after siRNA transfection. GAPDH was used as loading control. (B) Representative confocal images of cells labelled with Ki-67 staining (red) and DAPI (nucleus staining, blue) (scale bar = 50 μM). (C) Percentage of Ki-67-positive cells. (D) Steady-state mRNA level of p16^INK-4a and p21^WAF-1. GAPDH was used as a housekeeping gene. Results are expressed as fold induction in comparison to control fibroblasts at 24 hr. (E) Percentage of SA-βgal positive cells. (F) Steady-state mRNA level of Lamin B1. GAPDH was used as a housekeeping gene. (G) Steady-state mRNA level of IL-6, IL-8, MMP-1 and MMP-3. GAPDH was used as housekeeping gene. Results are expressed as fold induction in comparison to control fibroblasts at 24 hr. Statistical analyses were performed using an ANOVA II (*: p<0.05; **: p<0.01; ***: p<0.001).