The image contains multiple graphs and images illustrating the role of adenylate kinase (A k) in macrophage mitochondrial biogenesis following Listeria infection. A heatmap in panel A shows differential expression of the adenylate kinase (A K) gene family, with a scale of fold change (F C) on the right. The heat map shows the range of F C values of 9 A Ks from top to bottom, and the F C value decreases from A K 1 to A K 9. Bar graphs display R T-q P C R analysis of A k family m R N A expression in transgenic peritoneal macrophages (T G-p M a c s) with and without Listeria infection, and in Listeria-infected wild-type (W T) and A k 4 knockout (K O) T G-p M a cs. Graph B shows relative m R N A expression over A K 1 to A K 9, with the bars representing M O and Listeria infection. The bars show a fluctuating pattern, with highest expression at A K 6. Graph C shows relative m R N A expression over A K 1 to A K 9, with the bars representing W T and A K 4 superscript minus slash minus. The bars show a fluctuating pattern, with highest expression at A K 6. Two bar graphs labeled D compare intracellular A T P levels and A D P slash A T P ratios in W T and A k 4 superscript minus slash minus. For A T P levels, A K 4 shows highest value, while for A D P slash A T P ratio, W T shows the highest value. A Western blot image shows A k 4 protein levels in T G-p M a cs at various times post-infection. The A K 4 bands thickens over time, with a molecular mass of 26 k D. Beta-actin bands show the same thickness over time, with a molecular mass of 42 k D. A bar graph labeled F presents m t D N A copy numbers in Listeria-infected WT and A k 4 KO TG-p Macs at different time points. Another bar graph illustrates the expression of mitochondria-encoded genes in Listeria-infected W T and A k 4 superscript minus slash minus. The graph shows a general increasing pattern, with W T showing higher values compared to A K 4. A bar graph labeled G shows relative m R N A expression in 13 different mitochondrial-encoded genes. The bars represent W T and A K 4 superscript minus slash minus, with a fluctuating pattern; W T shows higher values than A K 4 superscript minus slash minus. Cryo-electron microscopy images depict mitochondria in Listeria-infected W T and A k 4 KO bone marrow-derived macrophages (B M D M s). In the left panel of W T, dark, irregular structures are enclosed in a red, dashed rectangle, while in the right panel, a close-up view of the irregular structure shows the central regions of the structures highlighted by red dots. In the left panel of A K 4 superscript minus slash minus, dark, elongated structures are enclosed in a red, dashed rectangle, while in the right panel, a close-up view of the elongated structure shows the central regions of the structures highlighted by red dots. A bar graph on the right shows mitochondria number per cell over W T and A K 4 superscript minus slash minus; W T shows higher value compared to A K 4 superscript minus slash minus. Two bar graphs labeled I and J show the mean fluorescence intensity (M F I) of MitoTracker Green and MitoTracker DeepRed in W T and A k 4 K O p M a cs, with W T showing higher value in both graphs. A line graph labeled K displays the oxygen consumption rate (O C R) in Listeria-infected W T and A k 4 K O p M a cs. The graph shows a general decreasing trend, with A K 4 showing lower values. Four bar graphs on the right show basal respiration, A T P production, proton leak, and maximal respiration for W T and A K 4 superscript minus slash minus; W T shows higher values in all the graphs.
Ak4 is essential for macrophage mitochondrial biogenesis after Listeria infection. (A) Heatmap illustrates differential expression of the Ak gene family in human monocyte-derived macrophages 24 h after Listeria infection (GSE34103). (B) RT-qPCR analysis of Ak family mRNA expression in TG-pMacs with or without Listeria infection (n = 4). (C) RT-qPCR analysis of Ak family mRNA expression in Listeria-infected WT and Ak4 KO TG-pMacs (n = 4). (D) WT and Ak4 KO pMacs were isolated 3 days after Listeria infection. Intracellular ATP levels (left) and ADP/ATP ratio (right) in WT (n = 6) and Ak4 KO (n = 7) pMacs were measured by bioluminescence. (E) Ak4 protein was determined in TG-pMacs after Listeria infection for the indicated time. (F) mtDNA copy number in Listeria-infected WT and Ak4 KO TG-pMacs was measured by qPCR at 0, 2, 6, 12, and 24 h after gentamicin treatment (n = 4). #, comparison with uninfected WT; $, comparison with uninfected Ak4 KO. (G) Mitochondria-encoded gene expressions in Listeria-infected WT and Ak4 KO TG-pMacs were analyzed by RT-qPCR at 24 h after gentamicin treatment (n = 3). (H) Representative images of Listeria-infected WT (n = 15) and Ak4 KO (n = 18) BMDMs were quantified at 24 h after infection using cryo-EM. Mitochondria were indicated by the red star. Scale bar, 1 μm or 200 nm. (I and J) MFI of MitoTracker Green (I) and MitoTracker Deep Red (J) measured mitochondrial mass and mitochondrial membrane potential, respectively, in WT and Ak4 KO pMacs, were analyzed by flow cytometry (n = 4). (K) OCR in Listeria-infected WT and Ak4 KO pMacs was measured using the Seahorse XF-96 analyzer (n = 3). mRNA expression levels were normalized to Actb, and protein levels were normalized to β-actin. mtDNA copy number was normalized to nDNA. Statistical significance was determined by an unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****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. Data are presented as mean ± SD. Source data are available for this figure: SourceData F2.