The image contains multiple graphs depicting the impact of A k 4 on mitochondrial D N A synthesis in macrophages after bacterial infection. Panel A shows Western blot results of P g c-1 alpha, T f a m, and alpha-tubulin protein levels in wild-type and A k 4 knockout bone marrow-derived macrophages with or without Listeria infection; P g c-1 alpha bands are darker compared to the bands of other two. Panels B and C display bar graphs measuring mitochondrial D N A copy number in scrambled and si T f a m-treated bone marrow-derived macrophages with or without A k 4 overexpression, and in wild-type and A k 4 knockout transgenic peritoneal macrophages pretreated with D M S O, deoxynucleotides, or gemcitabine, respectively, followed by Salmonella infection. Both graphs show a fluctuating pattern. Panels D through G present bar graphs showing the mean fluorescence intensity of MitoTracker Green and MitoTracker DeepRed in Salmonella-infected wild-type and A k 4 knockout transgenic peritoneal macrophages, analyzed by flow cytometry. All the graphs show a fluctuating pattern. Key findings include significant differences in mitochondrial D N A copy number and fluorescence intensity between wild-type and A k 4 knockout macrophages under various treatments.
Ak4 controls mtDNA synthesis through the deoxynucleotide metabolism in macrophages after bacterial infection. (A) Pgc-1α and Tfam protein levels in WT and Ak4 KO BMDMs with or without Listeria infection were determined by western blotting. (B) mtDNA copy number in scramble and siTfam-treated BMDMs with or without Ak4 overexpression was measured by qPCR (n = 4). (C–G) WT and Ak4 KO TG-pMacs were pretreated with DMSO, 10 μM dNs, or 5 μM Gem for 1 h, followed by infection with Salmonella at an MOI of 10 for 1 h. Cells were then treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for 24 h (C) or 6 h (D–G) prior to analysis. (C) mtDNA copy number in WT and Ak4 KO TG-pMacs was measured by qPCR (n = 4). (D–G) MFI of MitoTracker Green (D and E) and MitoTracker Deep Red (F and G) in Salmonella-infected WT and Ak4 KO TG-pMacs was analyzed by flow cytometry (n = 4). Protein expression levels were normalized to α-tubulin. mtDNA copy number was normalized to nDNA. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. *P < 0.05; **P < 0.01; ****P < 0.0001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. nDNA, nuclear DNA; MFI, mean fluorescence intensity. Source data are available for this figure: SourceData FS4.