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. 2024 Mar 11;15:2203. doi: 10.1038/s41467-024-46377-7

Fig. 4. Mitochondria sustain CD8+ T cell 3D motility by providing both ATP and mtROS.

Fig. 4

A–C Normalized 3D motility Score (A, n = 8 movies, ANOVA on Ranks with posthoc Student–Newman–Keuls’s), basal and CCCP-induced respiration (B, n = 3 independent biological samples, One-Way ANOVA with posthoc Holm Sidak’s) and normalized mtROS amount (MitoSox/FSC ratio; C, n = 7 independent biological samples, ANOVA on Ranks with posthoc Student–Newman–Keuls’s) of activated CD8+ T cells in the presence of the indicated doses of oligomycin. D, E Normalized 3D motility Score (D, n = 12 movies, unpaired two-tailed Student’s T-Test; E, n = 15 movies, ANOVA on Ranks with posthoc Tukey’s) and mtROS amount (MitoSox/FSC ratio; D, n = 6 independent biological samples, unpaired two-tailed Student’s T-Test; E, n = 6 independent biological samples, ANOVA on Ranks with posthoc Tukey’s) of activated CD8+ T cells in the presence of N-acetylcysteine (NAC) (in D) or MitoTEMPO (in E). F–P Normalized 3D motility Score (F, n = 16 movies, ANOVA in Ranks with posthoc Dunnet’s; J, n = 16 movies, ANOVA in Ranks with posthoc Dunnet’s; N, n = 9 movies, One-Way ANOVA with posthoc Holm Sidak’s vs ctrl), mtROS amount (MitoSox/FSC ratio; F, J, n = 7 independent biological samples, ANOVA in Ranks with posthoc Dunnet’s; N, n = 6 independent biological samples, One-Way ANOVA with posthoc Holm Sidak’s vs ctrl) and basal and ATP-linked respiration (G, K, n = 4 independent biological samples, unpaired Student’s T-Test; O, n = 4 independent biological samples, One-Way ANOVA on Repeated Measurements with posthoc Holm Sidak’s) of activated CD8+ T cells in the presence of the indicated doses of rotenone (F–K), antimycinA (antA) (J–M) or 3-nitropropionic acid (3-NP) (N–P). In (H, L, P), 3D motility was evaluated also in the presence of N-acetylcysteine (NAC) or oligomycin (oligo) (H–L, n = 12 movies, Two-Way ANOVA with posthoc Holm Sidak’s; P, 3-NP 10 µM, n = 12 movies, Two-Way ANOVA with posthoc Holm Sidak’s; oligo + 3-NP 1 mM, n = 12 movies, ANOVA on Ranks with posthoc Tukey’s; NAC + 3-NP 1 mM, n = 18 movies, ANOVA on Ranks with posthoc Tukey’s) and the relative (rotenone/control) or (antimycinA/control) changes in 3D motility Score for each condition were reported in (I, M) (for both n = 12 movies, ANOVA on Ranks with posthoc Dunnet’s). Schemes on the left in (F, J, N) indicate ETC complexes inhibited by each drug. Please note that the same controls were used for data in Figs. 4G and 4K since performed in the same experiments (data were split to improve clarity of the presentation). Data are expressed as mean ± SEM in (A–G, J, K, N, O), and as box plot (center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, 5th and 95th percentiles) in (H, I, L, M, P). Schemes in (F, J, N) have been created with BioRender.com.