Effect of MC1568 treatment on fibrogenesis in a CCl₄ induced fibrosis mouse model.

<p>Mice were CCl₄ treated twice a week for a total period of 4 weeks. After the second week, mice also received intraperitoneal injections with MC1568 (50 mg/kg) every two days for two more weeks. The day after the final CCl₄ injection, mice were sacrificed and liver tissue was extracted for analysis. (A) Sirius Red staining was performed to visualize deposited collagens. Image J software was used for quantification of the red surface area. Scale bar = 100 µm. (B) Serum levels of ALT and AST. (C) HDAC activity was measured in protein lysates of livers at the end of treatments using HDAC-Glo. (D) A mathematical correlation between the HDAC activity and the red stained area, after Sirius staining was determined using Pearson correlation method. The a and b refer to HDAC-activity values of the corresponding liver lysates of the images given for CCl₄ + MC1568 in A).</p

Class IIa and class IIb HDACs are constantly expressed during <i>in vitro</i> mouse hepatic stellate cell activation.

<p>Mouse HSCs were isolated from healthy mice and cultured for the indicated time. (A) Shows morphological changes associated to the mHSC activation process <i>in vitro</i>. (B) mRNA levels of activation markers <i>Col1a1</i>, <i>Col3a1</i>, <i>Acta2 and Lox</i> were determined using qPCR to confirm <i>in vitro</i> activation. (C) mRNA expression of Class IIa and IIb <i>Hdac</i>s was measured by qPCR. Values represent 3 replicates, ***: p<0,001, **: p<0, 01, *: p<0, 05.</p

Effect of MC1568 treatment on stellate cell activation markers.

<p>(A) Freshly isolated HSCs were plated in presence or absence of 1 µM MC1568 for 10 days. The effect on HSC activation markers <i>Acta2</i>, <i>Lox</i>, <i>Col1a1</i> and <i>Col3a1</i> was evaluated by qPCR. (B) Mouse HSCs were cultured for 10 days in presence or absence of 1 µM MC1568. The effect on HSC activation markers Collagen I, Lysyl oxidase and α-Smooth muscle actin was investigated by western blot. β-actin was used as a loading control. (D) The effect of MC1568 treatment on HSC proliferation was investigated with an EdU incorporation assay. Cells were cultured for 48 hours in the presence or absence of MC1568. Nuclei were stained with 4′,6-Diamidino-2-phenylindole. The percentage of EdU-positive cells was determined from three independent experiments. (D) In order to test the reversibility of MC1568 treatment, freshly isolated mouse HSCs were treated for 7 days, after 7 days the inhibitor was washed out and cells were further cultured until day 10 (recovery). Then cells were collected and mRNA expression of HSC activation markers was determined. (E) Freshly isolated mouse HSCs were cultured for seven days. At day seven, cells were formalin fixed and stained with an antibody against the acetylated form of tubulin. Prior to fixation, cells were treated with 1 µM MC1568 for 24 hours. Nuclei were visualized with 4′,6-Diamidino-2-phenylindole. Scale bar = 100 µM. * p<0.05, ** p<0,01.</p

List of antibodies for immunocytochemistry and western blot.

<p>List of antibodies for immunocytochemistry and western blot.</p

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.</p