Research Paper Volume 12, Issue 16 pp 16621—16646
Prolonged treatment with Y-27632 promotes the senescence of primary human dermal fibroblasts by increasing the expression of IGFBP-5 and transforming them into a CAF-like phenotype
Figure 2. The inhibition of ROCK signaling promotes the cellular senescence of HDFs. (A) HDFs were treated with PBS or 10 μM Y-27632, then were collected at the indicated times, stained with annexin V-FITC combined with PI, then analyzed by FACS for apoptotic cells. (B) HDFs were treated with PBS (upper) or 10 μM Y-27632 (lower), then fixed at the indicated times and analyzed using a SA-β-gal staining kit for senescent cells. scale bar = 100 μm. (C) Quantification of (B), the percentage of SA-β-gal positive cells (blue, arrows) was calculated based on counting a total of 500 cells. (D) HDFs were transfected with siRNAs for ROCK1 and/or ROCK2, or with a scrambled siRNA as a control (sictrl), and at 48 h after transfection, cells were fixed and stained with SA-β-gal for senescent cells (blue, arrows), scale bar = 100 μm. (E) Quantification of SA-β-gal-positive cells in (D), the percentage of SA-β-gal-positive cells was calculated based on counting a total of 500 cells. All experiments were performed at least 3 times,*P<0.05, **P<0.01, ***P<0.005 when two groups were compared as indicated or were compared to the corresponding controls.