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. 2021 Feb 24;6(1):e00036-21. doi: 10.1128/mSphere.00036-21

FIG 3.

FIG 3

Treatment of infected macrophages with DP (ZBTB25 inhibitor) and CI994 (HDAC1 inhibitor) enhanced the intracellular clearance of M. tuberculosis. THP-1-derived macrophages were infected with M. tuberculosis H37Rv. After M. tuberculosis infection, cells were treated with DP (20 μM) and/or CI994 (15 μM) or rifampicin (1 μg/ml) for 24 h. (A) Intracellular bacterial viability was determined based on the number of CFU. Isolation of bacilli from the macrophages was carried out at 24 h. The number of viable bacilli in each of the plate was assayed by plating lysed macrophages on 7H10 agar plates and incubating the plates at 37°C for 3 weeks and counting the CFU. Values are shown from 3 independent experiments (mean ± SD). *, survival is significantly different from infected samples (IF); P ≤ 0.05. (B) qPCR for the expression of the IL-12B mRNA transcript (normalized to β-actin mRNA expression). *#, IL-12B expression is significantly different from uninfected (UIF) and infected samples (IF), respectively; P ≤ 0.05. (C) ELISA of IL-12p40 after inhibitor treatment. *#, IL-12p40 levels are significantly different from uninfected (UIF) and infected samples (IF), respectively; P ≤ 0.05. (D) DP treatment 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 PCR. (E) Immuno-cytochemical imaging shows HDAC1 does not colocalize with ZBTB25 inside the macrophage nucleus after DP treatment. (F) Intracellular viability of M. tuberculosis in infected PBMC after treatment with DP, CI994, and rifampicin was determined by counting the number of CFU. *, survival is significantly different from infected sample (IF); P ≤ 0.05.