Fig. 5: MARCH5 prevents prolonged type І IFN signaling in murine macrophages stimulated by LPS.

a RT-qPCR analysis of mIFNB1 mRNA levels in RAW 264.7 cells harvested after 4 h of treatment with 200 ng/ml LPS. Data are means ± SD of n = 4; one-tailed ratio unpaired Student’s t-test with CI = 95%, P values are shown at the top of the graph. b Experiment as in Fig. 6a. Western blot analysis of pHSPA8 and pJNK, cGAS and STING. c, RT-qPCR analysis of mIFNB1 mRNA in RAW 264.7 cells pretreated with 12.5 μM NU7026, 3 μM AZD7648, and 80 μM VBIT-12 as indicated and treated with 200 ng/ml LPS. Data are means ± SD of n = 4; one-way ANOVA with CI = 95% and Bonferroni’s post hoc test. d Experiment as in Fig. 6c. Western blot analysis of pHSPA8. e RT-qPCR to measure mIFNB1 mRNA levels after harvest of WT and March5 KO RAW 264.7 cells treated with LPS for the indicated times. Normalized expression Data are means ± SD of n = 4 independent experiments; two-way ANOVA with CI = 95% and Bonferroni’s post hoc test. f Experiment as in Fig. 6e. Western blot analysis of pHSPA8 and pJNK. g RT-qPCR to measure mIFNB1 mRNA levels after harvest of March5 KO RAW 264.7 cells transfected with simDNA-PKcs for 72 h and treated with LPS for the indicated times. Normalized expression data are the mean ± SD of n = 4 independent experiments. Two-way analysis of variance with CI = 95% followed by Bonferroni post hoc test. h, The cell lysates were prepared as described in 6g. Western blot analysis of pHSPA8 and pJNK. Each experiment was performed at least three times. ***P ≤ 0.005.