FIG 2.

Knocking down of ZBTB25 enhances IL-12p40 and reduces the intracellular survival of M. tuberculosis. THP-1 macrophage cells were transfected with ZBTB25 siRNA, or scrambled siRNA control using HiPerFect transfection reagent. (A) Western blot analysis of macrophage cells in which the expression of endogenous ZBTB25 was knocked down using ZBTB25 siRNA. Lane 1, normal macrophages; lane 2, macrophages transfected with scrambled siRNA control; lane 3, macrophages transfected with ZBTB25 siRNA. (B) Immuno-cytochemical staining with anti-ZBTB25 antibody and DAPI of control THP-1 macrophages. Cells in which ZBTB25 was knocked down showed reduced fluorescence compared to the control macrophages. (C) Knocking down of ZBTB25 blocks the recruitment of the HDAC1 silencing complex to the IL-12B promoter. Status of (i) ZBTB25, (ii) HDAC1, and (iii) Sin3a on the IL-12B promoter by ChIP followed by PCR. UIF, normal macrophages; IF, macrophages transfected with scrambled siRNA and infected with M. tuberculosis; IF+siRNA, macrophages transfected with ZBTB25 siRNA and infected with M. tuberculosis. (D) IL-12B expression is upregulated in M. tuberculosis-infected macrophages upon ZBTB25 inhibition. qPCR of IL-12B when ZBTB25 is knocked down. *#, IL-12B expression is significantly different from uninfected (UIF) and infected (IF) samples; P ≤ 0.05. (E) ELISA of IL-12p40 when ZBTB25 is knocked down in macrophages. *, IL-12p40 levels are significantly different from infected (IF) sample; P ≤ 0.05. (F) Survival of M. tuberculosis decreases in macrophages when ZBTB25 is knocked down. Macrophages in which ZBTB25 was knocked down were infected with M. tuberculosis. Macrophages not treated with siRNA but infected with M. tuberculosis were also kept as control. Intracellular bacterial viability was determined by counting the number of CFU. Data shown here are the mean ± SD of three independent experiments. *, survival is significantly different from both controls; P ≤ 0.05.