Figure 6.

Increased AKT activity in MKP-1^−/− macrophages is controlled by p38-induced miR-21 expression. (A) Phospho-AKT (P-AKT)–positive macrophages were counted from immunostained serial cryosections of gastrocnemius muscles obtained from WT and MKP-1^−/− mice postinjury (P.I.; see pictures in Fig. S5 D). (B) Gene expression analysis by RT-PCR in FACS-isolated macrophage populations at 6 d after injury from mice treated with wortmannin or vehicle for the preceding 3 d. (C) WT and MKP-1^−/− BM macrophages were stimulated with LPS at the indicated times. Western blotting analysis using phospho-AKT and AKT antibodies. (D) Expression of miR-21 was analyzed by qPCR in isolated macrophage populations at 3 and 6 d after injury from WT and MP-1^−/− mice or WT mice treated with SB203580 (SB). (E) Total number of macrophages per milligram of muscle at 6 d after injury. Muscles were injected with scrambled or mimic miR-21 oligonucleotides at 3 d after injury. (F) Primary macrophages were transfected with scrambled or ant–miR-21 oligonucleotides and stimulated with LPS for the indicated times; IL-1β or IL-10 expression analysis. (G) As in F, primary macrophages were transfected with scrambled (Scr) or miR-21 mimic oligonucleotides. (H) Primary macrophages infected with a retrovirus expressing MKK6E or an empty retrovirus (C, control), in the absence or presence of SB203580, were analyzed for MKK6, phospho-p38 (P-p38), phospho-ATF2 (P-ATF2), and p38 expression by Western blotting (left) or for miR-21 mRNA expression by qPCR analysis (right). Means ± SEM of at least three experiments. ^# or ***, P < 0.001; **, P < 0.01; *, P < 0.05.