We used an RNAi-based knockdown approach to understand whether MAT genes play a role during HSC activation and proliferation. As shown in Fig. 3A,B, knockdown of MAT2A for 48 hours in primary rat HSCs resulted in decreased HSC activation as measured by the lower levels of Col1A2 mRNA and type I collagen protein, respectively, compared to scrambled RNAi controls. A similar decrease in α-SMA mRNA and protein was also observed after MAT2A silencing. MAT2A gene silencing did not cause any change in cellular proliferation at this time, thereby indicating a lack of any toxicity at the 48-hour timepoint during which gene expression changes were measured (Fig. 3C). However, longer periods of MAT2A knockdown

(80 hours) resulted in decreased BrDU incorporation in HSCs indicative of suppressed cell growth (Fig. Torin 1 research buy 3C). Despite several attempts, we were unable to get sufficient knockdown of the MAT2β gene in primary rat HSCs. In order to examine the role of MAT2β in HSC activation, we performed gene silencing studies in the easily transfectable, human LX-2 cell line. As shown in Fig. 4A, knockdown of either MAT2A by 80% or MAT2β by 93% inhibited expression of Col1A2 and α-SMA mRNA by 50% as compared 3-MA mouse to scrambled RNAi-treated cells. These results were further confirmed at the protein level (Fig. 4B). Concurrent with the findings in primary HSCs (Fig. 3), knockdown of MAT2A or

MAT2β in LX-2 cells did not significantly affect cell growth up to 48 hours but there was decreased proliferation at the 72-hour timepoint (Fig. 4C). Toxicity effects of MAT gene knockdown were also evaluated by performing apoptosis assays in LX-2 cells. As shown in Fig. 4D, knockdown of either MAT2A or MAT2β

up to 48 hours did not significantly affect the number of apoptotic cells compared to scrambled RNAi controls. However, at 72 hours there was a significant increase in the percent apoptosis when compared to scrambled RNAi. Because MAT2A or MAT2β silencing affects growth in LX-2 cells, we investigated 上海皓元 the mechanism by which these genes influence cell proliferation and apoptosis. Our results showed that MAT2A knockdown lowered intracellular SAMe levels by 75% compared to control or scrambled RNAi-treated cells (Table 4). We also examined the effect of MAT2A or MAT2β silencing on the survival signaling pathways, ERK and PI-3K, in LX-2 cells. In Fig. 5 we showed that knockdown of MAT2A did not significantly influence phosphorylation of ERK or AKT (a measure of PI-3K signaling) but knockdown of MAT2β prevented activation of both ERK and AKT in LX-2 cells, thereby indicating a role of this gene in signal transduction pathways associated with HSC activation. The expression of MAT genes in the liver is a determinant of cell proliferation and differentiation. Although MAT1A is a marker of differentiation, MAT2A and MAT2β are markers of growth and dedifferentiation.