Figure 4.
Actin assembly in ETC protein depleted cells. (A) Mitochondrial polarization in MEFs (± SD) after 0.2 μg/ml ethidium bromide(EtBr)/50 μg/ml uridine treatment. Ctrl, untreated. Uridine, uridine treatment alone (10 d). CCCP - 10 min 20 µM CCCP-treated Ctrl cells. Circles indicate individual cell measurements (n ≥ 86 cells per group combined from two independent experiments). Experiments done in 25 mM glucose with serum. (B) MEFs under uridine alone or ethidium bromide/uridine treatment (EtBr) for 10 d, stained for actin filaments (green) and mitochondria (red). Scale bars: 5 μm. (C) % cells (± SD) displaying actin assembly after time in ethidium bromide/uridine, with and without 100 µM CK666. n ≥ 98/15 cells/fields of view (FOV) per group combined from two experiments. Experiments done in 25 mM glucose with serum. (D) Lactate production (± SD) in ethidium bromide cells (4 d, EtBr-4; 10 d, EtBr-10) and uridine-treated control (10 d), with and without 100 µM CK666 after 6 h. Points indicate individual well measurements starting with 75,000 cells/well. n = 8 individual well measurements from four independent experiments. n.s. P > 0.05; **** P < 0.0001. Statistical significance was calculated by one-way ANOVA using Tukey’s multiple comparisons test. Experiments done in 2 mM glucose without serum. (E) WT and NDUFS4 KO MEFs stained for actin filaments (green) and mitochondria (red). Scale bars: 5 μm. (F) % cells (± SD) displaying actin assembly in WT and NDUFS4 KO MEFs, with and without 10 min of 100 µM CK666 treatment. n ≥ 70/12 cells/FOVs per group combined from three independent experiments. * P = 0.018. Statistical significance was calculated using unpaired two-tailed t tests. Experiments done in 25 mM glucose with serum. (G) Cytosolic ATP levels in WT or NDUFS4 KO MEFs upon 100 µM CK666 treatment. n ≥ 30 cells per group combined from three independent experiments. Arrow indicates drug treatment. Experiments done in 25 mM glucose with serum. (H) Lactate production (± SD) in WT and NDUFS4 KO cells, with and without 100 µM CK666 after 6 h. Points indicate individual well measurements starting with 75,000 cells/well. n = 8 individual well measurements from four independent experiments. n.s. P > 0.05; **** P < 0.0001. Statistical significance was calculated by one-way ANOVA using Tukey’s multiple comparisons test. Experiments done in 2 mM glucose without serum. (I) % cells (± SEM) displaying actin assembly in control or Leigh syndrome patient fibroblasts. n ≥ 76/13 cells/FOVs per group combined from two independent experiments. Experiments done in 25 mM glucose with serum. Statistical tests are tabled in Fig. S10 B. Number of experiments, FOV, sample sizes, and statistical tests are provided in Table S1.

Actin assembly in ETC protein depleted cells. (A) Mitochondrial polarization in MEFs (± SD) after 0.2 μg/ml ethidium bromide(EtBr)/50 μg/ml uridine treatment. Ctrl, untreated. Uridine, uridine treatment alone (10 d). CCCP - 10 min 20 µM CCCP-treated Ctrl cells. Circles indicate individual cell measurements (n ≥ 86 cells per group combined from two independent experiments). Experiments done in 25 mM glucose with serum. (B) MEFs under uridine alone or ethidium bromide/uridine treatment (EtBr) for 10 d, stained for actin filaments (green) and mitochondria (red). Scale bars: 5 μm. (C) % cells (± SD) displaying actin assembly after time in ethidium bromide/uridine, with and without 100 µM CK666. n ≥ 98/15 cells/fields of view (FOV) per group combined from two experiments. Experiments done in 25 mM glucose with serum. (D) Lactate production (± SD) in ethidium bromide cells (4 d, EtBr-4; 10 d, EtBr-10) and uridine-treated control (10 d), with and without 100 µM CK666 after 6 h. Points indicate individual well measurements starting with 75,000 cells/well. n = 8 individual well measurements from four independent experiments. n.s. P > 0.05; **** P < 0.0001. Statistical significance was calculated by one-way ANOVA using Tukey’s multiple comparisons test. Experiments done in 2 mM glucose without serum. (E) WT and NDUFS4 KO MEFs stained for actin filaments (green) and mitochondria (red). Scale bars: 5 μm. (F) % cells (± SD) displaying actin assembly in WT and NDUFS4 KO MEFs, with and without 10 min of 100 µM CK666 treatment. n ≥ 70/12 cells/FOVs per group combined from three independent experiments. * P = 0.018. Statistical significance was calculated using unpaired two-tailed t tests. Experiments done in 25 mM glucose with serum. (G) Cytosolic ATP levels in WT or NDUFS4 KO MEFs upon 100 µM CK666 treatment. n ≥ 30 cells per group combined from three independent experiments. Arrow indicates drug treatment. Experiments done in 25 mM glucose with serum. (H) Lactate production (± SD) in WT and NDUFS4 KO cells, with and without 100 µM CK666 after 6 h. Points indicate individual well measurements starting with 75,000 cells/well. n = 8 individual well measurements from four independent experiments. n.s. P > 0.05; **** P < 0.0001. Statistical significance was calculated by one-way ANOVA using Tukey’s multiple comparisons test. Experiments done in 2 mM glucose without serum. (I) % cells (± SEM) displaying actin assembly in control or Leigh syndrome patient fibroblasts. n ≥ 76/13 cells/FOVs per group combined from two independent experiments. Experiments done in 25 mM glucose with serum. Statistical tests are tabled in Fig. S10 B. Number of experiments, FOV, sample sizes, and statistical tests are provided in Table S1.

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