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 6. Y-27632 induces expression of IGFBP-5 in HDFs in vitro and in vivo. (A) Genes whose expression levels changed differently at 12 h and 48 h in (Figure 4) were detected by RT-qPCR analysis. Fold mRNA expression levels were normalized by the control at the indicated times. (B) HDFs were treated with PBS and 10 μM (y+) Y-27632 at the indicated times and total RNAs were extracted for RT-qPCR analysis of IGFBP-5. The fold change of IGFBP-5 relative to the control (expression level as 1) is shown. (C) HDFs were transfected with siRNAs specific for ROCK1 and/or ROCK2, with a scrambled siRNA as a control, and at 48 h after transfection, the mRNA expression level of IGFBP-5 was determined by RT-qPCR analysis. The fold change of IGFBP-5 in ROCK knockdown cells relative to the control (expression level as 1) is shown. (D) HDFs were treated with PBS or 10 μM (y+) Y-27632 and the conditioned medium was collected at the indicated times for ELISA analysis of IGFBP-5 protein level. (E) HDFs were transfected with siRNAs specific for ROCK1 and/or ROCK2, with a scrambled siRNA as a control, and at 48 h after transfection, the cells were replaced with fresh medium and cultured for another 48 hours to collect the conditioned medium for ELISA analysis of IGFBP-5 protein level. (F) The reconstituted skin formed from grafting HDFs together with human epidermal cells were collected after treatment with 10 μM Y-27632 or PBS for 2 weeks and total RNAs were extracted from the dissected dermis of the skin for RT-qPCR analysis of human IGFBP-5. All experiments were repeated at least 3 times. * P<0.05,**P<0.01, *** P<0.005 when compared to the control.