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. 2019 Apr 23;87(5):e00809-18. doi: 10.1128/IAI.00809-18

FIG 9.

FIG 9

Proinflammatory response by VpOmpU involves TLR-mediated p38 and JNK MAP kinase activation. (A and B) Increased phosphorylation of p38 and JNK in RAW 264.7 (A) and THP-1 (B) cells in response to VpOmpU as evidenced by immunoblotting and densitometric analysis of the bands. Whole-cell lysates were analyzed for the phosphorylation of p38 and JNK at different times following VpOmpU treatment. Total p38 and JNK in the cells were also determined. Densitometric analysis of the immunoblots confirmed increased phosphorylation of p38 and JNK in RAW 264.7 and THP-1 cells in response to VpOmpU. For the densitometric analysis, the band intensities of p-p38 or p-JNK in the samples were calculated as those above the band intensities of p38 or JNK, respectively, and fold changes upon VpOmpU treatment were estimated with respect to the buffer-treated cells. Results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus band intensities in the buffer-treated cells]). (C) Decrease in TNF-α in response to VpOmpU upon pretreatment with inhibitors of the MAP kinases p38 (VX745) and JNK (JNK-IN8) in RAW 264.7 and THP-1 cells. Results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus cells treated with VpOmpU only]). (D and E) Increased phosphorylation of p38 and JNK in VpOmpU-treated BMDMs from wild-type (WT) mice but no change in phosphorylation status of p38 and JNK in VpOmpU-treated BMDMs from TLR2−/− mice. Densitometric analysis of the immunoblots shows increased phosphorylation of JNK at 15 min and of p38 at both 15 min and 30 min in the wild-type BMDMs and no change in the phosphorylation status of JNK and p38 in the TLR2−/− BMDMs in response to VpOmpU. For the densitometric analysis, the band intensities of p-p38 or p-JNK in the samples were calculated as those above the band intensities of p38 or JNK, respectively, and fold changes upon VpOmpU treatment were estimated with respect to the buffer-treated cells. Results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus band intensities in buffer-treated cells]). β-Actin and GAPDH were used as the loading controls in the immunoblots of the whole-cell lysates.