Figure 5.
LPA promotes mitochondrial fusion via MTCH2.(A) MitoTracker red staining of WT cells treated with vehicle or the DGAT1 inhibitor T863 (20 µM) and DGAT2 inhibitor PF-06424439 (20 µM; together, DGATi) for 16 h. DGATi-treated cells were then incubated with cycloheximide (CHX; 10 µM) or starved in HBSS for 5 h in the presence of inhibitor. Scale bars = 5 µm. (B) Quantification of mitochondrial morphology in cells described in A. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (C) MitoTracker red staining of WT cells treated with vehicle or the GPAT inhibitor FSG67 (GPATi; 75 µM) for 16 h. GPATi-treated cells were incubated with CHX (10 µM) or starved in HBSS for 5 h with or without BSA (0.1% wt/vol) in the presence of inhibitor. Scale bars = 5 µm. (D) Quantification of mitochondrial morphology in cells described in C. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (E) Top: Schematic representation of content-mixing in vitro mitochondrial fusion assay with addition of LPA. Bottom: In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol (cyto) or the indicated amount of LPA 16:0. Data are presented as fusion efficiency normalized to the control reaction. Error bars show mean + SEM of three or four independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (F) In vitro fusion efficiency of mitochondria isolated from WT or MTCH2−/− cells with or without addition of LPA 16:0 (400 µM) or PA (400 µM). Data are presented as fusion efficiency normalized to the WT control reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (G and H) In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol from cells treated with vehicle, ACCi (2 µg/ml; G), or GPATi (75 µM; H) for 16 h. Data are presented as fusion efficiency normalized to the no-cytosol reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (I) Schematic representation of the TAG synthesis pathway and the generation of LPA stimulating MTCH2-dependent mitochondrial fusion.

LPA promotes mitochondrial fusion via MTCH2. (A) MitoTracker red staining of WT cells treated with vehicle or the DGAT1 inhibitor T863 (20 µM) and DGAT2 inhibitor PF-06424439 (20 µM; together, DGATi) for 16 h. DGATi-treated cells were then incubated with cycloheximide (CHX; 10 µM) or starved in HBSS for 5 h in the presence of inhibitor. Scale bars = 5 µm. (B) Quantification of mitochondrial morphology in cells described in A. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (C) MitoTracker red staining of WT cells treated with vehicle or the GPAT inhibitor FSG67 (GPATi; 75 µM) for 16 h. GPATi-treated cells were incubated with CHX (10 µM) or starved in HBSS for 5 h with or without BSA (0.1% wt/vol) in the presence of inhibitor. Scale bars = 5 µm. (D) Quantification of mitochondrial morphology in cells described in C. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated between hyperfused values by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (E) Top: Schematic representation of content-mixing in vitro mitochondrial fusion assay with addition of LPA. Bottom: In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol (cyto) or the indicated amount of LPA 16:0. Data are presented as fusion efficiency normalized to the control reaction. Error bars show mean + SEM of three or four independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (F) In vitro fusion efficiency of mitochondria isolated from WT or MTCH2−/− cells with or without addition of LPA 16:0 (400 µM) or PA (400 µM). Data are presented as fusion efficiency normalized to the WT control reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (G and H) In vitro fusion efficiency of mitochondria isolated from WT cells with or without addition of cytosol from cells treated with vehicle, ACCi (2 µg/ml; G), or GPATi (75 µM; H) for 16 h. Data are presented as fusion efficiency normalized to the no-cytosol reaction. Error bars show mean + SEM of three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s HSD test. *, P < 0.05. (I) Schematic representation of the TAG synthesis pathway and the generation of LPA stimulating MTCH2-dependent mitochondrial fusion.

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