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. 2023 Jan 17;120(4):e2212813120. doi: 10.1073/pnas.2212813120

Fig. 4.

Fig. 4.

Antimicrobial effects of HDAC7 require PPP- and NADPH oxidase-dependent ROS. (A) NADPH/NADP+ ratio in the indicated BMM populations infected with EC958. (B) Measurement of real time OCR in the indicated BMM populations infected with MG1655 (Left). Area under the curve analysis until 15 min of OCR determinations was calculated with post-background correction (Right). (C–E) Measurement of total ROS in the indicated BMM populations (C and D) or RAW264.7 (E), pretreated with cM-H2DCFDA (10 μM) for 30 min before infecting them with MG1655 for 30 min. Representative flow cytometry plot (Left), as well as relative median fluorescence intensity (MFI) data (Right), are shown for (C and D). (F) Relative intramacrophage EC958 loads at 2 h p.i. in indicated BMM populations, pretreated with the NOX2 inhibitor GSK2795039 (GSK, 10 μM) for 1 h. (G) Relative intramacrophage bacterial loads of EC958 or EC958katG at 2 h p.i. in the indicated BMM populations. Graphical data (mean ± SEM, n = 3) are combined from three independent experiments and are normalized to the Hdac7+/+ uninfected control (C), uninfected MacBlue control (D), uninfected empty vector control (E), or MacBlue control infected with wild type EC958 (F and G). Statistical significance was determined using repeated measure two-way ANOVA followed by Sidak’s multiple comparison test [A and B (Left)–G] or unpaired t test with Welch’s correction [B (Right)] (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).