Skip to main content
. 2018 Feb 8;3(3):e98268. doi: 10.1172/jci.insight.98268

Figure 9. BMDM responsiveness, polarization, and cytokine expression after exogenous TNF-α stimulation.

Figure 9

(A) Western blotting showed that iRhom2-deficient (iRhom2–/–) bone marrow–derived macrophages (BMDMs) treated with 20 ng/ml TNF-α display activation of TNF-α downstream signaling protein phospho-NF-κB (p-NF-κB), while wild-type BMDMs displayed increased levels of p-NF-κB and p-JNK. Wild-type and iRhom2–/– samples were run in the same gel but were noncontiguous. (B) Scanning densitometry shows significant increases in p-NFκB and p-JNK relative to the loading control GAPDH. Data are shown as the mean ± SEM, n = 3 for each experimental group.*P ≤ 0.05, Student’s t test. TNF-α–stimulated iRhom2–/– BMDMs show significantly reduced (C) M1 and (D) M2 marker expression compared with controls, while (E) M1 and (F) M2 cytokine expression was partially affected. Gene expression normalized to respective stimulations without TNF-α treatment. Flow cytometric analysis of iRhom2–/– BMDMs: (G) A contour plot of the number of macrophages expressing TNFRI (MFI: 5,737 ± 59) and/or TNFRII (MFI: 14,615 ± 112) compared with wild-type BMDMs (TNFRI MFI: 3,830 ± 340; TNFRII MFI: 8,456 ± 251), and (H) biological replicates were quantified. Data are shown as the mean ± SEM, n = 4 for each experimental group. **P ≤ 0.01, 1-way ANOVA and post-hoc test. MFI, mean fluorescence intensity; TNFRI, type I TNF receptor; TNFRII, type II TNF receptor.