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. 2020 Jul 22;9:e55415. doi: 10.7554/eLife.55415

Figure 1. SIRT2 aids in mycobacterial survival ex vivo.

Figure 1.

(A) Analysis of microarray data from Mehta et al., 2016 reveals differential expression of SIRT1, SIRT2, HDAC6 and HDAC9 in THP1 cells 24 hr pi. FC: Fold Change in the gene expression in infected THP1 cells as compared to uninfected control, t: moderated t-statistics, B: B-statistics or log-odds that the gene is differentially expressed, adj.p.val: p-value after adjustment for multiple testing. (B) Mouse peritoneal macrophages were infected with Mtb laboratory strain H37Rv for 24 hr and SIRT2 expression was assessed through RT-PCR. (C–D) Mouse peritoneal macrophages infected with Mtb for 24 hr were stained with anti SIRT2 antibody followed by FACS analysis. (E and F) Mouse peritoneal macrophages, pre-treated with AGK2 for 2 hr, were infected with GFP expressing H37Rv and maintained in 10 µM of AGK2 for 48 hr. (E) Percentage of infected cells as analyzed by flow cytometry. (F) In a parallel experiment, cell lysates were plated for bacterial CFU at 0 hr and 48 hr pi. (G–I) RAW 264.7 macrophages were transfected with shRNAs specific for SIRT2. (G) 48 hr post transfection, SIRT2 protein levels were assessed by western blot. Transfected cells were infected with GFP expressing H37Rv for 48 hr. (H) Percentage of infected cells as analyzed by flow cytometry at 48 hr pi. (I) In a parallel experiment, cell lysate was plated for enumeration of CFU. (J–M) Representative dot plots and bar graphs to show the percentage of necrotic and apoptotic cell populations in the uninfected (J and K) and Mtb infected (L and M) peritoneal macrophages with or without AGK2 treatment as analyzed by FACS analysis. (N and O) Representative overlay plots and quantification of cellular ROS measured by staining cells with CellROX followed by flow cytometry. Mouse peritoneal macrophages treated with AGK2 (10 µM), NAC (10 mM) or both were infected H37Rv for 48 hr. (P and Q) Representative overlay plots and bar graph depicting cellular ROS in these cells 48 hr pi. (R) In a parallel experiment, cell lysates were plated for CFU enumeration. Data shown is representative of at least two independent experiments performed in triplicates. The data values represent mean ± SD (n = 3). *p<0.05, **p<0.005, ***p<0.0005.

Figure 1—source data 1. SIRT2 inhibition enhances anti-mycobacterial potential of host macrophages.
Source data for Figure 1B, D–F, H, I, K, M, O, Q and R.