Role of class II HDACs during HSC activation by selective siRNA mediated knock-down.

<p>Freshly isolated HSCs were transfected with siRNA after 24 hours of culture and a second time on the fifth day of culture, RNA was collected 3 days after the final transfection (day 8). (A) <i>Hdac</i> knock-down was evaluated by qPCR. (B) In addition the effect on activation markers was determined by qPCR. The dashed line represents the gene expression level in cells transfected with non-specific siRNA. (C) At day 4 after the second transfection protein samples were harvested. The effect of HDAC knock-down on Lox and expression was confirmed by WB. β-actin was used as loading control. (D) The effect of HDAc inhibition by MC1568 treatment was investigated after 10 days of treatment in culture. Total RNA was isolated from cultured cells, a microRNA reverse transcription was performed followed by qPCR assay for selective amplification of miR-29a, miR-29b and miR-29c, and RNU6 was used as internal control. (E) Freshly isolated HSCs were transfected with siRNA after 24 hours of culture and a second time on the fifth day of culture, RNA was collected 3 days after the final transfection (day 8). Total RNA was isolated from cultured cells and a qPCR assay for selective amplification of miR-29a, miR-29b and miR-29c was performed, RNU6 was used as internal control. miR-29 expression in HDAC knock down samples was calculated relative to miR-29 expression in a sample transfected with non-targeting siRNA (represented by dashed line). Graphs are representative of at least three experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.