Figure 2

cANGPTL4 enhanced fibroblast proliferation and migration and reduced fibroblast collagen expression. (a) Representative FACs scatter plots of fibroblasts subjected to 24 h of the indicated treatments, then pulsed with 40 μM BrdU for 30 min prior to FAC S analysis. Fibroblasts were harvested and fixed in 70% ice-cold ethanol, their DNA was denatured with 1.5 M HCl, rinsed to neutralize acidity, then stained with FITC-conjugated anti-BrdU antibody and propidium iodide according to manufacturer’s protocol. Cell populations in the G0/G1, S and G2 phases are gated and the percentages are indicated in parentheses (b,c) Representative images and measurements of percentage wound closure by fibroblast migration. Human primary dermal fibroblasts (1 × 10⁴ cells per insert) were seeded into silicone inserts (ibidi, USA) on a 24-well culture plate (Corning Life Sciences, USA) and allowed to attach overnight in a humidified incubator with 5% CO₂ at 37 °C before the removal of the inserts. Fibroblasts were then pre-treated with mitomycin-C (1 mg/mL of culture media) for 2 h to induce cell cycle arrest prior to the start of the scratch assay. Culture medium was replaced with fresh media containing the indicated treatments. The plate was then placed into the Cell-IQ machine for real-time imaging. Wound closure rate was assessed using the on-board analysis software that measured the percentage change in surface area occupied by fibroblasts. (d) Relative mRNA expression of COL1 and COL3 in fibroblasts treated for 24 h with vehicle (saline) or 12 μg/mL cANGPTL4 in the presence or absence of 10 ng/mL TGFβ. Ribosomal protein L27 was used as housekeeping gene for normalization of target gene expression. (e) Relative protein expression of COL1A2, COL3A1 and phospho-Smad3 in fibroblasts treated for 24 h with vehicle (saline) or 12 μg/mL cANGPTL4 in the presence or absence of 10 ng/mL TGFβ. Bars represent mean densitometry measurements for COL1A2 and COL3A1 (normalized against β-tubulin), and phospho-Smad3 (normalized against total Smad3). Loading controls were obtained from the same sample lysates. Images are representative of 5 fields of view from n = 5 independent experiments; values are means ± SD, n = 5 independent experiments. **P < 0.01 (Mann-Whitney U-test).