FIGURE 1.

Effects of siRNAgp46 on gp46 expression, collagen secretion, and proliferation of aHSCs. A, aHSCs were treated with siRNAgp46 at 2, 5, and 10 nm in DMEM containing 2% FBS for 72 h. aHSCs were also treated with siRNAgp46 at 10 nm for 1–5 days. The dose- and time-dependent suppression of gp46 expression was analyzed by Western blotting. B, collagen secretion from siRNAgp46-treated aHSCs in culture medium containing 2% FBS was assayed at day 3 of transfection. The amount of collagen in four samples was quantified by spectrophotometry at 540 nm, and the absolute concentrations were deduced from the standard curve of rat tail collagen provided by the assay kit and expressed as mean ± S.E. *, p < 0.05 compared with siRNAGFP. C, aHSCs treated with siRNAgp46 at 10 nm in DMEM with 2% FBS for 1–5 days were stained with antibody against αSMA-Cy3 and DAPI. The number of cells was monitored by counting the stained cells in 10 random fields per slide. Data are mean ± S.D. of three independent experiments. *, p < 0.05 compared with siRNAGFP. D, aHSCs treated with siRNAgp46 at 10 nm in DMEM with 2% FBS for 1–5 days were stained for apoptotic change (TUNEL positivity) and for autophagic change with antibody against LC3B. The apoptotic and autophagy-induced cells were counted in 40 random fields/slide. Data are mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 compared with siRNAGFP. E, representative photomicrographs of aHSCs undergoing apoptosis at day 5 of transfection. Arrowheads indicate TUNEL-positive cells. Scale bars = 100 μm. F, expression of caspase 3 and cleaved caspase 3 was analyzed for aHSCs treated with 10 nm of siRNAgp46 in DMEM containing 2% FBS for 3 days to examine the effect of siRNAgp46 on apoptosis induction. G, quantitative analyses of levels of cleaved caspase 3 protein in untreated and gp46 siRNA-treated aHSCs. The results are mean ± S.D. of three samples. *, p < 0.05.