The image contains multiple graphs illustrating the impact of A k 4 gene deletion on mitochondrial function in mice following Listeria infection. Panel A shows a schematic representation of the C R I S P R slash C a s 9 system used to generate A k 4 knockout mice. Panel B presents a bar graph comparing relative m R N A expression levels of A k 4 in M O, wild-type (W T), and A k 4 knockout (K O) bone marrow-derived macrophages (B M D M s) stimulated with L P S slash I F N. W T shows the highest value. Panel C displays a Western blot analysis of A k 4 protein levels in W T and A k 4 K O B M D Ms. The bands of A K 4 are faded compared to the bands of beta-actin. Panels D and E show bar graphs of the mean fluorescence intensity (M F I) of MitoTracker Green and MitoTracker DeepRed, respectively, in W T and A k 4 K O B M D Ms with and without Listeria infection; both graphs show a fluctuating pattern with W T showing the highest value. Panel F is a line graph depicting oxygen consumption rates (O C R) measured by Seahorse analysis in W T and A k 4 K O B M D M s under different conditions. The graph shows a fluctuating pattern, with W T showing the highest value. Panel G includes bar graphs of various mitochondrial respiration parameters, such as basal respiration, A T P production, proton leak, maximal respiration, and spare capacity. The graphs for basal respiration and A T P production show a decreasing pattern, while the graphs for proton leak, maximal respiration, and spare capacity show a fluctuating pattern. Panel H shows a bar graph of the M F I of MitoSox in W T and A k 4 K O B M D M s with and without Listeria infection. The graph shows a fluctuating pattern. The data are presented as mean plus or minus standard deviation, with statistical significance indicated by asterisks.
Ak4 is essential for maintaining mitochondrial number and function after Listeria infection. (A) Schematic representation of the generation of conventional Ak4 KO mice using the CRISPR/Cas9 system. (B and C) Ak4 mRNA (B) and protein (C) levels in LPS/IFNγ-stimulated WT and Ak4 KO BMDMs were analyzed by RT-qPCR and western blotting, respectively (n = 3). (D–H) WT and Ak4 KO BMDMs were infected with or without Listeria for 6 h. (D and E) MFI of MitoTracker Green (D) and MitoTracker Deep Red (E) were analyzed by flow cytometry (n = 4). (F and G) OCR levels were measured by Seahorse analysis (n = 3). (H) MFI of MitoSox was analyzed by flow cytometry (n = 4). mRNA levels were normalized to Actb. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (B and D–H). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. MFI, mean fluorescence intensity. Source data are available for this figure: SourceData FS3.