Figure 6

PARP-1/AIF pathway is involved in BCG-induced RAW264.7 cell necrosis. RAW264.7 cells were treated with indicated conditions for 24 h, the cells were then collected for immunobotting analysis or flow cytometric analysis. (A) Immunoblotting analysis for PARP-1 and its downstream mediator AIF proteins in RAW264.7 cells treated with indicated conditions for 24 h. Both Wnt3a-CM and PARP-1 inhibitor 3-AB displayed an ability to down-regulate PARP-1 and AIF. (B) NAD⁺ analysis for NAD⁺ in RAW264.7 cells treated with indicated conditions for 12 h. Wnt3a could inhibit the NAD⁺ depletion of RAW264.7 cells infected with BCG. (C) Flow cytometric analysis showed a time-dependent inhibition of BCG-induced necrosis mediated by Wnt3a or PARP-1 inhibitor 3-AB (2.5 mmol/L), suggesting the PARP-1 was involved in BCG-induced necrosis for RAW264.7 cells, and the Wnt signaling could inhibit RAW264.7 cell necrosis by down-regulation of PARP-1 activity. (D) Representatives of scatter dot plot from three independent experiments of flow cytometric analysis of the necrotic fraction of RAW264.7 cells treated with indicated conditions for 6 h. Compared to a control-CM treated cells * or #: p < 0.05, ** or ##: p < 0.01. Control-CM treated cells compared to its corresponding Wnt3a-CM treated cells, #: p < 0.05; ##: p < 0.01. Data represented the mean ± SD from three independent triplicate experiments (N = 9).