Figure S1.
NME3 acts downstream of PLD6 for mitochondrial clustering. (A) Effects of PLD6-EGFP overexpression on mitochondria. Wild-type or catalytic dead (H156N) PLD6-EGFP were co-transfected with mito-BFP into HeLa cells. While PLD6-EGFP expression results in mitochondrial clustering, the expression of PLD6H156N-EGFP lead to mitochondrial fragmentation. Scale bar, 10 μm. (B) Knockdown efficiency of siNME3. Due to the lack of a specific antibody for NME3, we generated HA knock-in NME3 HeLa cell to monitor the effect of NME3 depletion by siRNA SMARTPool (Dharmacon). The knock down efficiency was measured by Western blotting with anti-HA antibody, indicated by arrow, and normalized with tubulin. Star indicates a non-specific signal. Comparable expression of PLD6-mCherry was observed in control and NME3 knockdown cells. (C) NME3 is required for the mitochondrial clustering effect induced by PLD6-mCherry overexpression. Control or NME3 knockout mouse embryonic fibroblasts were transfected to overexpress mCherry, EGFP, PLD6-mCherry, or NME3-GFP. Scale bar, 10 μm. Bar for inset images, 2 μm. (D) The mitochondria in these cells were labeled by mito-BFP, and the average area of individual mitochondrium was measured by ImageJ and shown in D. Each dot represents the averaged mitochondrium area of one cell. (E and F) Rescuing ability of NME3-GFP in siNME3 cells. Wild-type NME3-GFP was transfected into siNME3 HeLa cells. The rescuing abilities of these constructs were demonstrated by the significant restoration of the average size of mitochondria shown in F. Scale bar, 10 μm. Bar for inset images, 2 μm. (G) The expression level of different NME3-GFP constructs. HeLa cells transfected with indicated NME3-GFP constructed were blotted with anti-GFP antibody. Due to its high expression level, less cell lysate of NME3ΔN-GFP transfected cell was loaded in doer to avoid saturated signal of GFP. Data are expressed as mean ± SD of three independent experiments and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Source data are available for this figure: SourceData FS1.

NME3 acts downstream of PLD6 for mitochondrial clustering. (A) Effects of PLD6-EGFP overexpression on mitochondria. Wild-type or catalytic dead (H156N) PLD6-EGFP were co-transfected with mito-BFP into HeLa cells. While PLD6-EGFP expression results in mitochondrial clustering, the expression of PLD6H156N-EGFP lead to mitochondrial fragmentation. Scale bar, 10 μm. (B) Knockdown efficiency of siNME3. Due to the lack of a specific antibody for NME3, we generated HA knock-in NME3 HeLa cell to monitor the effect of NME3 depletion by siRNA SMARTPool (Dharmacon). The knock down efficiency was measured by Western blotting with anti-HA antibody, indicated by arrow, and normalized with tubulin. Star indicates a non-specific signal. Comparable expression of PLD6-mCherry was observed in control and NME3 knockdown cells. (C) NME3 is required for the mitochondrial clustering effect induced by PLD6-mCherry overexpression. Control or NME3 knockout mouse embryonic fibroblasts were transfected to overexpress mCherry, EGFP, PLD6-mCherry, or NME3-GFP. Scale bar, 10 μm. Bar for inset images, 2 μm. (D) The mitochondria in these cells were labeled by mito-BFP, and the average area of individual mitochondrium was measured by ImageJ and shown in D. Each dot represents the averaged mitochondrium area of one cell. (E and F) Rescuing ability of NME3-GFP in siNME3 cells. Wild-type NME3-GFP was transfected into siNME3 HeLa cells. The rescuing abilities of these constructs were demonstrated by the significant restoration of the average size of mitochondria shown in F. Scale bar, 10 μm. Bar for inset images, 2 μm. (G) The expression level of different NME3-GFP constructs. HeLa cells transfected with indicated NME3-GFP constructed were blotted with anti-GFP antibody. Due to its high expression level, less cell lysate of NME3ΔN-GFP transfected cell was loaded in doer to avoid saturated signal of GFP. Data are expressed as mean ± SD of three independent experiments and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Source data are available for this figure: SourceData FS1.

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