Extended Data Figure 9: JNK signalling is required for mitochondrial fragmentation induced by C18:0 removal.

a, Treatment of HeLa cells with 1 µM gambogic acid does not induce apoptosis. 10 μM gambogic acid was used as a positive control for apoptosis induction, assessed by cleaved caspase-3 levels. 1 µM gambogic acid neither induces caspase cleavage (shown here) nor causes cells to die (data not shown). Cells were treated with 10 µM gambogic acid for 1 h, or for all other concentrations for 3 h. See Supplementary Fig. 20 for image of the uncropped full western blot. b, Activation of TFR1 by treating cells with 1 µM gambogic acid leads to mitochondrial fragmentation that is reversed by 1 h C18:0 pre-treatment. Representative images are shown here and quantification of mitochondrial fragmentation is shown in Fig. 3i (n = 15). c, Treatment of HeLa cells with C18:0 to inhibit TFR1 causes reduced JNK signalling activity, assayed by p-JNK levels on an immunoblot. See Supplementary Fig. 20 for image of the uncropped full western blot. d, Removal of C18:0, as well as treatment with gambogic acid, induces shuttling of phosphorylated JNK into the nucleus. Cells were stained with phospho-JNK antibody (left) and relative levels of nuclear to cytosolic phospho-JNK signal was quantified (right) (n = 37 cells). ***P < 0.001, two-tailed t-test. Error bars show s.d. e, JNK signalling is required for TFR1 activation to induce mitochondrial fragmentation. HeLa cells were treated with the JNK inhibitor SP600125 30 min before gambogic acid treatment to activate TFR1. Representative images are shown here and quantification of mitochondrial fragmentation is shown in Fig. 3j (n = 15).