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. 2022 Sep 19;13(5):e01086-22. doi: 10.1128/mbio.01086-22

FIG 4.

FIG 4

Treatment with methylglyoxal (MGO) hampers control of M. tuberculosis infection and HIF-1-regulated responses of BMM A-C.BMM were incubated with 200 μM MGO and infected with BCG 4 h after. (A to C) The mean fold increase of vegfa (A), il1b (B), and inos (C) mRNA was measured in triplicate BMM cultures ± SEM as measured by RT-PCR. Differences are significant at *, P ≤ 0.05 and **, P ≤ 0.01, one-way ANOVA with Welch’s adjustment. (D) The concentration of nitrite in the supernatants of MGO-treated and/or BCG-infected BMM was measured 72 h after culture. (E to G) BMM incubated with 125 μM MGO for 4 h and infected with M. tuberculosis thereafter. The mean fold increase of vegfa (E), il1b (F), and inos (G) mRNA was measured in triplicate BMM cultures ± SEM was measured by RT-PCR. Differences between infected groups are significant at *, P ≤ 0.05 and **, P ≤ 0.01, one-way ANOVA with Welch’s adjustment. (H) The lactate concentration in the supernatants from BMM treated with MGO 4 h before infection with M. tuberculosis was measured by lactate dehydrogenase assay. The mean lactate concentration ± SEM in BMM supernatants 72 h after infection is shown. Differences are significant at *, P ≤ 0.05 and **, P ≤ 0.01, one-way ANOVA with Welch’s adjustment. (I) A group of BMM was treated with IFN-γ starting 48 h before M. tuberculosis-gfp infection. IFN-γ-treated and control BMM were treated with 400 μM MGO 4 h before infection with M. tuberculosis-gfp (MOI 1:1). (I to K) Representative histograms (I), the mean percentage of infected BMM (J), and the MFI of intracellular M. tuberculosis (K) quantified 5 days after infection are shown. Differences are significant at *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001, one-way ANOVA with Welch’s correction. (L and M) Representative micrographs (L) of uninfected MGO-treated and control BMM and the mean ± SEM intensity of HIF-1α immunolabeling (M) of MGO-treated, uninfected-, or M. tuberculosis-infected BMM determined 24 h after infection. Differences are significant at *, P ≤ 0.05 and ***, P ≤ 0.001, one-way ANOVA with Welch’s adjustment. (N) The relative HRE activity in RAW macrophages treated or not with MGO was evaluated 24 h after BCG infection. The mean relative luciferase levels ± SEM in triplicate independent cultures are depicted. Differences are significant at **, P ≤ 0.01, one-way ANOVA with Welch’s correction.