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. 2015 Aug 26;56(9):5656–5669. doi: 10.1167/iovs.15-17204

Figure 6.

Figure 6

Hic-5 induces fibrogenic activity in HTM cells. (A) Immunoblotting of cell lysates (72 hours post infection) derived from serum-starved HTM cells expressing adenovirus-encoded recombinant Hic-5 showed a significant increase in α-SMA and collagen-1 compared to control cells infected with adenovirus encoding DsRed (n = 4, mean ± SD). *P < 0.05. (B) HTM cells transfected with Hic-5 siRNA (72 hours post transfection) exhibit a decrease in Hic-5 expression (green immunofluorescence staining) and actin stress fibers (rhodamine-phalloidin labeling in red) compared to cells transfected with scrambled siRNA (siSc). Scale bar: 50 μm. (C) HTM cells transfected with Hic-5 siRNA show a significant decrease in Hic-5 (>80%), α-SMA, and collagen-1 compared to cells treated with siSc based on immunoblot analyses. β-actin was probed as a loading control. The histograms show a densitometry-based representation of the decrease in the levels of Hic-5, α-SMA, and collagen-1 proteins (values are mean ± SD, n = 4, *P < 0.05). (D) Knockdown of Hic-5 expression in HTM cells suppresses TGF-β2-induced expression of αSMA and collagen-1. Serum-starved HTM cells treated with TGF-β2 (8 ng/mL for 24 hours) exhibited significant increase in expression of Hic-5, αSMA, and collagen-1 by immunoblotting analysis. These effects of TGF-β2 were suppressed in cells transfected with Hic-5 siRNA. Histograms represent the densitometry-based values from an average (mean ± SD) of four independent experiments. *P < 0.05. β-actin was immunoblotted as loading control.