Differences between groups were rated significant at a probability error (P) of less than 0.05. We evaluated cell proliferation in nontransduced (C), rAd.A20, rAd.Nter, rAd.7n, and rAd.βgal-transduced NMuLi cells. This cell line responds in a physiologic manner to growth factor-induced cell cycle progression.15 Overexpression of A20 increased by 1.6-fold cell counts/well when compared to C and rAd.βgal transduced cells, 24 hours after addition of 10% FBS, (Fig. 1A, n = 4-6; P < 0.05). Similarly, rAd.Nter and rAd.7Zn-transduced cells showed a 1.8- and 1.9-fold increase, respectively, in cell counts/well (Fig. 1A; n = 3-4; P < 0.05 versus C and P < 0.01 versus

rAd.βgal). This indicated that independent overexpression of Nter or 7Zn increases proliferation in NMuLi cells. We reproduced these results in HepG2 selleck chemical cells, validating their use in subsequent experiments (Fig. S2A; n = 4; P < 0.001). We previously reported that A20′s pro-proliferative effect in hepatocytes related, at least in part, to decreased p21 expression.15 We confirmed in HepG2 that overexpression of full-length A20, but not Nter or 7Zn, significantly decreased p21 messenger RNA (mRNA) levels as compared to

β-gal-expressing cells (Fig Selleckchem MAPK Inhibitor Library 1B; n = 3-5; P < 0.05). As for NF-κB inhibition17 (Fig. 1C; n = 3), cooperation between Nter and 7Zn domains was required to decrease p21, signifying that other mechanism(s) must account for their independent pro-proliferative effect in hepatocytes. Given potential discrepancies between cell lines and primary cells, we validated these results in MPH: full-length A20 but neither 7Zn nor Nter decreased p21 mRNA levels (Fig.

S2B; n = 2; P < 0.05), or inhibited TNF-induced IκBα degradation (Fig. S2C; n = 3). Since IL-6 is central to hepatocyte proliferation, we measured IL-6 levels in supernatants of C, rAd.A20, MCE rAd.Nter, rAd.7Zn, and rAd.βgal transduced HepG2 stimulated with TNF (200 U/mL) and LPS (10 μg/mL) for 6 hours to mimic the physiologic triggers of IL-6 secretion after hepatectomy.1 IL-6 levels significantly increased in all groups following TNF/LPS, as compared to corresponding nonstimulated cells (6.5- to 9.9-fold, Fig. 2A). However, IL-6 levels were significantly lower in supernatants of A20 overexpressing HepG2 compared to all other groups (Fig. 2A; n = 4-7; P < 0.01 versus C; and P < 0.001 versus rAd.Nter, rAd.7Zn, and rAd.βgal). This result indicates that IL-6 transcription partially relies on NF-κB.22 Notably, neither Nter nor 7Zn decreased TNF/LPS-induced IL-6 secretion, with 7Zn overexpression moderately, yet significantly increasing it (Fig. 2A; P < 0.01 versus C). Despite lower IL-6 levels in supernatants of A20-overexpressing cells, STAT3 phosphorylation, downstream of IL-6, was enhanced at baseline (∼150-fold; P < 0.001), 6 hours (P < 0.001) and 24 hours (P < 0.