Fig. 6: Phosphorylated NDRG1^Ser336 interacts with CDC42 to drive mitochondrial fission.

a, The log₂-transformed fold change (FC) of interaction of Flag–NDRG1^WT or Flag–NDRG1^Ser336Ala with CDC42, ARHGAP35 and ARHGEF10 (n = 3 independent experiments). b, Pulldowns of Flag (using Flag M2 agarose) and immunoblots and quantification for GFP and Flag levels in OA-treated (2.5 h) NIH3T3 cells co-expressing Flag–NDRG1^WT or Flag–NDRG1^Ser336Ala and GFP–CDC42^WT (n = 3 independent experiments). Flag-tagged empty vector is the negative control. Ponceau is loading control. Quantification for relative enrichment of GFP in Flag pulldowns was calculated by normalizing the densitometric value of GFP to the densitometric value of Flag. c, Representative live-cell imaging of mCherry–NDRG1^WT and MitoTracker green in siCon or siCdc42 NIH3T3 cells. Magnified insets are shown. Orange arrowhead: NDRG1 (mCherry) mediating fission. White arrowhead: mCherry/MitoTracker reflecting NDRG1/mitochondrial co-localization before fission. Yellow arrowheads: divided mitochondria after fission. Please refer to Supplementary Video 12 (siCon cells; mCherry–NDRG1^WT) and Supplementary Video 13 (siCdc42 cells; mCherry–NDRG1^WT). d, Graphical representation for duration of interaction between mCherry–NDRG1 and mitochondria (MitoTracker) in siCon or siCdc42 cells, and whether interactions lead to division or are futile. e, Quantification for mean duration of mCherry–NDRG1^WT/mitochondria (MitoTracker) interaction is shown (siCon 6 cells and siCdc42 11 cells from n = 3 independent experiments; each tracked cell was monitored on an independent plate). f, Representative confocal images of NIH3T3 cells transfected with indicated siRNAs and cultured in serum-free medium with MitoTracker green for 30 min. Magnified insets are shown. Quantifications for mitochondria number and mitochondrial size/shape descriptors are shown (siCon 142 cells, siRictor 105 cells, siNdrg1 91 cells, siCdc42 64 cells, siMff 106 cells, siDnm1l 128 cells, siOpa1 86 cells and siMfn1 83 cells from n = 8 (siCon), n = 6 (siRictor and siMff), n = 5 (siNdrg1, siOpa1 and siMfn1), n = 4 (siCdc42) and n = 7 (siDnm1l) independent experiments). Grey areas indicate mitochondria fission-deficient models. g, MitoTracker CMXRos fluorescence in siCon and siCdc42 cells cultured in serum-free medium in presence of OA for 5 h (siCon 102 cells and siCdc42 116 cells from n = 3 independent experiments). Individual replicates and means are shown. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, two-tailed unpaired Student’s t-test (a, b, e and g); one-way ANOVA and Dunnett’s multiple comparisons test (f). Please refer to Supplementary Table 10 statistical summary, and Supplementary Table 8. Source numerical data are available in Source Data Extended Data Table 1, and unprocessed blots are available in the Source Data for this figure.

Source data