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 1. Inhibition of ROCK signaling decreases the growth of HDFs. (A) One x 106 HDFs were seeded in growth medium in the presence of 10 μM Y-27632 or PBS (as control); cells were then collected at the indicated times to analyze cell numbers using an automatic cell-counter; (B) HDFs were cultured under the same conditions as in (A); cells were collected at the indicated times to analyze cell proliferation using a CCK8 kit, and the percentage of cell growth was calculated relative to the corresponding control (PBS) as 100. (C) and (D): HDFs were cultured with 10 μM Y-27632 or control PBS, then fixed at the indicated times and stained with the proliferation marker EdU (green) and DAPI to stain nuclei (blue). The quantification of EdU-positive cells is shown in (C), which was calculated as the percentage of green cells (total count of 200 cells) of DAPI positive cells (blue); representative images of EdU staining are shown in (D). Scale bars = 100 μm. (E) HDFs were transfected with siRNAs specific for ROCK1 (siROCK1), ROCK2 (siROCK2) or both ROCK1 and ROCK2 (siROCK1+2); control HDFs were transfected with a scrambled siRNA (sictrl), and cells were collected at 48 h after transfection to detect the expression of ROCK1 and ROCK2 by western-blot (E). (F) One x 106 HDFs were transfected with siRNAs specific for ROCK1 and/or ROCK2 and a scrambled siRNA (sictrl), at 12 and 48 h after transfection cells were collected to analyze cell proliferation using a CCK-8 kit. The percentage of cells was calculated relative to cells transfected with the negative control siRNA as 100. (G) One x 106 HDFs were transfected with siRNAs specific for ROCK1 and/or ROCK2 and a scrambled siRNA (sictrl) plus/minus Y-27632 and at 48 h after transfection, cells were collected to analyze cell proliferation using a CCK-8 kit. The percentage of cells was calculated relative to cells transfected with the negative control siRNA without treatment of Y-27632 as 100. All experiments were repeated at least 4 times. *P<0.05, **P<0.01, when two groups were compared as indicated, or were compared to the corresponding controls.