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. 2021 Dec 8;12:7115. doi: 10.1038/s41467-021-27426-x

Fig. 3. Macrophages in conditions of lactic acidosis display mitochondrial depolarization and characteristics typical of starving cells.

Fig. 3

a–k Monocytes were polarized into macrophages in the absence (Mφ) or presence of lactic acid (LA-Mφ), lactic acid and acetoacetate (LA-Mφ + AcAc), sodium lactate (Lactate-Mφ), or under acidosis (HCl-Mφ). a, b Cells with depolarized mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry using MitoTracker Green and MitoTracker Deep Red probes at the indicated time point (a) (mean ± SD; n = 4, two-way ANOVA followed by a Tukey post hoc test was performed for statistical analysis) or at day 3 (b) (mean ± SD; n = 14 for Mφ; n = 13 for LA-Mφ; n = 5 for HCl-Mφ; n = 4 for Lactate-Mφ). Representative dot plots on day 3 (a, right panel). c Macrophages with or without depolarized mitochondrial membrane potential (“Depol” and “Pol” populations, respectively) were isolated from day 3 LA-Mφ by flow cytometry with MitoTracker Green and MitoTracker Deep Red probes. Purity was >99%. d Intracellular ATP levels were measured in a semiquantitative assay. The results were normalized against the Mφ condition for each donor (n = 5). e Levels of total AMPKα, AMPKα phosphorylated on Thr172 (pAMPKα) and HSC-70 were analyzed by Western blotting at 6 h (representative images from four independent experiments); the values correspond to pAMPKα/HSC-70 and pAMPKα/AMPKα band intensity ratios. f Intracellular acetyl-CoA levels determined on day 3 (n = 4). g–j Cell size was measured on day 3 (g–j) and day 7 (i), by determining the mean ± SD of the diameter of Mφ in each population (g, n = 5) or relative cell size by flow cytometry with the FSC-A parameter (i, n = 7). h Photomicrographs of the Mφ population on day 3 (representative images from five independent experiments); size bar, 10 µm. j Representative dot plots from one donor of FSC-A measurement. a–j The boxplots display a median line, interquartile range (IQR) boxes, min to max whiskers; a two-tailed Mann–Whitney U test was performed for statistical analysis unless specified otherwise. Source data are provided in a Source Data file.