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. 2015 Sep 24;5:13730. doi: 10.1038/srep13730

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Copyright © 2015, Macmillan Publishers Limited

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(a) Casp3 activity was assessed in liver protein extracts from mice treated 3 days with methylene blue (3 mg/kg) or vehicle and then injected with LPS/GALN or PBS. Data represent the mean and SEM of 8 independent experiments. Statistical differences were assessed with one-way ANOVA (p < 0.0001) and a post hoc Dunnett’s test comparing all conditions to LPS/GALN-treated conditions ***p < 0.001. (b) Proteasomal nLPnLDase activity measured in liver protein extracts from mice treated with methylene blue and/or LPS/GALN in the presence or absence of 0.1 μM proteasome inhibitor epoxomicin. Data represent the mean and SEM of 3 independent experiments. No statistical differences were observed. (c) RCasp3 activity after treatment with DMSO, 1 μM of the pan-caspase inhibitor Q-VD-OPh, or 0.1 μM of the reversible proteasome inhibitor bortezomib. (d) DEVDase activity in liver protein extracts from mice treated with or without methylene blue and LPS/GALN after addition of DMSO, 1 μM Q-VD-OPh, or 0.1 μM bortezomib. Data represent the mean and SEM of 3 independent experiments. Statistical differences were assessed in a one-way ANOVA (p < 0.0001) followed with a post hoc Dunnett’s test comparing all conditions to DMSO in control-treated mice (*). DEVDase activity in DMSO treated protein extracts from LPS/GALN (no methylene blue)-treated mice did not change significantly in the presence of bortezomib. (e) Western blot of liver protein extracts from two different mice per group showing levels of proCasp3 and cleaved-Casp3 (p17/p19). (f) Western blot of liver protein extracts from two different mice per group showing levels of cleaved β actin and β-actin. (g) Quantitative analyses of cleaved β-actin over full-length actin levels in liver protein extracts from mice treated with methylene blue or vehicle in the presence or absence of LPS/GALN. Data represent the mean and SEM of 8 independent experiments. Statistical evaluations were conducted as described in a.