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 7. Y-27632 promotes the senescence of HDFs through regulation of GATA4/IGFBP-5. (A) HDFs were treated with or without 10 μM Y-27632 or with or without 100 ng/ml IGFBP-5 for 48 h, then were fixed and analyzed using a SA-β-gal staining kit to detect senescent cells. scale bar = 100 μm. (B) 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. (C) HDFs were transfected with a siRNA targeting IGFBP-5 (siIGFBP-5) or a scrambled siRNA (sictrl). After transfection for 24 h, HDFs were treated with PBS or 10 μM Y-27632 for 48 h and were then fixed and analyzed using a SA-β-gal staining kit to detect senescent cells. scale bar = 100 μm. (D) Quantification of SA-β-gal-positive cells in (C), the percentage of SA-β-gal-positive cells was calculated based on counting a total of 500 cells. (E) HDFs were cultured in growth medium in the presence of PBS or 10 μM Y-27632, then collected at 48 h after treatment for RT-qPCR analysis of GATA4. The fold change of GATA4 in Y-27632 treated HDFs relative to the control (expression level as 1) is shown. (F) HDFs were transfected with siRNAs for ROCK1 and/or ROCK2, or a scrambled siRNA as a control, and at 48 h after transfection, the mRNA expression level of GATA4 was detected by RT-qPCR analysis. The fold change of GATA4 in the ROCK knockdown HDFs relative to the control (expression level as 1) is shown. (G) HDFs were transfected with a scrambled siRNA (sictrl), siRNAs targeting GATA4 (siGATA4), together with either PBS or 10 μM Y-27632 or 100 ng/ml IGFBP-5 and 10 μM Y-27632 alone for 48 h, then were fixed and analyzed using a SA-β-gal staining kit to detect senescent cells, scale bar = 100 μm. (H) Quantification of SA-β-gal-positive cells in (G), the percentage of SA-β-gal-positive cells was calculated based on counting a total of 500 cells. (I) Proposed scheme indicating the mechanism of ROCK regulating the senescence of human skin dermal cells. All experiments were repeated at least 4 times, * P<0.05,**P<0.01, *** P<0.005, when two groups were compared as indicated, or were compared to the corresponding control.