Figure 6

rhPN does not affect HGF proliferation nor modulate myofibroblast differentiation in vitro. (A) HGFs cultured on collagen or collagen + rhPN coated plates for 1, 3, 5, 7, and 9 days were assessed for proliferation using CyQUANT assay kit to determine DNA contents. A standard curve was used to extrapolate cell number. Data represents fold cell number increase ± s.d. relative to day 1. Data was analyzed via two-way ANOVA d (p > 0.05). (B) HGFs cultured on collagen or collagen + rhPN coated plates for 1 day and 7 days were assessed for gene expressions of ACTA2. Target gene expression was normalized to 18 S using the ΔΔCt method. Data represents mean fold gene expressions ± s.d. relative to control day 1 (collagen alone) of 3 independent experiments in triplicates. Data was analyzed via Student’s t-test (unpaired) within each time-point. (C) Western blot was used to assess α-SMA protein level of HGFs cultured on collagen or collagen + rhPN coated plates. GAPDH was used as a loading control. (D) Immunocytochemistry was performed with HGFs cultured on collagen or collagen + rhPN coated plates for 7 days to visualize myofibroblasts. Representative fluorescent images of HGFs labeled for α-SMA (green), F-actin (red), and nuclei (blue). Myofibroblasts were revealed by α-SMA-positive stress-fibers. Scale bar, 50 μm. (E) Fixed collagen gel contraction assay of HGFs in the absence and presence of rhPN (5 μg/ml). Gel contraction was quantified by loss of gel weight, compared with gels lacking cells. Data was analyzed via Student’s t-test (unpaired) within each time-point (p > 0.05; ns, not significant).