Fig. 5

IL-16 production repressed Rev-erb? expression in macrophages response to mycobacterium challenge. A and B Monocyte-derived macrophages were pretreated with or without neutralized antibody anti-IL-16 (1 µg/mL) or rhIL-16 protein (10 ng/mL) for 18 h. Then, the cells were infected with M. marinum (MOI = 5:1) for 4 h. Afterward, the cells were washed to remove unphagocytosed bacteria and further incubated with or without blocking antibodies (1 µg/mL) or rhL-16 (10 ng/mL) for additional time. Immunofluorescence by confocal imaging was used to detect the nuclear expression of Rev-erb? (A) and LXR4 (B) at 3 dpi. C Western blotting analysis was used to examine the expression of Rev-erb?, according to A. D WT or Il-16-/- BMDMs were infected with H37Rv (MOI = 3:1) for 4 h, washed to remove unphagocytosed bacteria and incubated for additional time with or without rhL-16 (10 ng/mL). Rev-erb? protein level was determined by Western blotting at 3 dpi. E Immunofluorescence was used to detect the nuclear expression of pro-IL-16 in monocyte-derived macrophages infected with M. marinum (MOI = 3:1) at 3 dpi by confocal analysis. F A bioinformatic search revealed a putative GAPBA? response motif flanked by a 12 bp sequence of TCTCTCCCGGTC in the Rev-erb? promoter. Ch-IP, re-ChIP and PCR assay were performed in macrophages infected with M. marinum (MOI = 5:1) at 3 dpi. Normal anti-IgG (2 ?g), anti-pro-IL16 (2 ?g), anti-GAPB? (2 ?g) and anti-HDAC3 (2 ?g) Abs were used for each immunoprecipitation. PCRs with non-immunoprecipitated genomic DNA (Input) were also performed as control. G Relative level of PCR- amplified Rev-erb? DNA by Image J, according to F. *p<0.05 compared to media, Student's t-test. The graph shown is representative of 2 independent experiments.