FIGURE 2. Both RNase and deubiquitinase activities of MCPIP are required for M2 polarization.

The peritoneal macrophages isolated from C57BL/6 wild-type mice were transfected with either control vector, or MCPIP expression plasmid, or expression vectors for D141N mutant, or Dub mutant for 48 hr. Forced expression of MCPIP induced expression of M2 markers, Arg1 and FIZZ1 (A, B, C, D). Expression of M2 markers (Arg1, FIZZ1), assayed by qRT-PCR (A, B) and immunoblot (C, D), were inhibited by loss of deubiquitinase or RNase activity of MCPIP. ROS production, assayed by DHR 123 (E), ER stress markers GRP78 and IRE-1, assayed by qRT-PCR (F, G) and autophagy as measured by expression of Beclin-1 and LC3II assayed by qRT-PCR, and immunoblot (H, I), were inhibited by loss of either catalytic activity of MCPIP. ^*P < 0.05 vs EV (empty vector); ^#P <0.05 vs MCPIP. Experiments were repeated three times. Six different fields containing at least 200 cells were analyzed for ROS production.