Figure 4

Effect of CCN2 in sustaining HSC survival. Freshly isolated rat HSC were cultured in 6-well plates in 5% FBS DMEM for 24 h, followed by serum deprivation for 48 h. The cells were cultured for another 24 h in the absence or presence of 100 ng/ml CCN2 ("CCN2"). In the CCN2 protection assay, the cells were incubated with 10 μM Bay11-7082 for 24 h alone ("Bay11") or following pre-treatment of the cells with CCN2 for 1 h ("CCN2+Bay11"). For the Bay11 blocking assay, the cells were pre-treated with Bay11-7082 for 30 min and cultured for another 24 h in the presence of 100 ng/ml CCN2 ("Bay11+CCN2"). At the end of incubation time period, (A) cells were trypsinized and survival was determined by Trypan blue exclusion, or (B) cell viability was also quantified by measurement of the fluorescence intensity using CellTiter-Glo™ reagent, or (C) cell apoptosis was assessed by measurement of caspase-3 activity at 405 nm using a luminescence assay kit. *P < 0.05 vs. control; ^#P < 0.05 vs. CCN2 group; **P < 0.01 and * P < 0.05 vs. Bay11-7082 group.