Figure S4.
PLD6 is dispensable for the steady mitochondrial targeting of full-length NME3. (A) Effect of PLD6 depletion on the subcellular localization of endogenous NME3. HeLa cells with GFP knock-in to the C-terminus of NME3 were transfected with PLD6 siRNA SMARTPool. (B and C) The knock-down and mitochondrial localization efficiency of endogenous NME3-GFP were quantified by Western blotting, confocal microscopy, and ImageJ analysis. (D) Effect of PLD6 depletion on the subcellular localization of exogenous NME3. NME3-GFP was transfected into control or PLD6-depleted HeLa cells. (E and F) The knock-down and mitochondrial localizing efficiency of ectopic NME3-GFP were quantified by Western blotting, confocal microscopy, and ImageJ analysis. (G and H) Subcellular distribution of NME3. HeLa cells with GFP or HA knock-in to the C-terminus of NME3 were transfected with control or PLD6 siRNA SMARTPool. After 72 h, cells were subjected to fractionation to measure the mitochondrial targeting ratio of endogenous NME3 with two different tags. Similar to their distribution observed under fluorescent microscopy, the amount of NME3-GFP or NME3-HA in mitochondrial fraction was not decreased by the depletion of PLD6. (I) Localization of ALPS-NME3-EGFP. The chimeric NME3-EGFP with its N17 replaced by the amphipathic helix lipid packing sensor (ALPS) derived from the PA-binding domain of Spo20 (62–79 amino acid) was expressed in HeLa cells and imaged together with mito-BFP. (J) The enrichment of NME3-GFP at mitochondrial contact interface prior to fusion. The dynamic distribution of NME3-GFP in two contact mitochondria was observed in Cos-7 cells co-transfected with NME3-GFP and Tom20-mCherry, which labels mitochondrial outer membrane. Arrows indicate the contact interface between two closely positioned mitochondria. Scale bars, 10 μm. Bars for inset images, 2 μm. Data are expressed as mean ± SD of three independent experiments and analyzed with Student’s t test. **P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS4.

PLD6 is dispensable for the steady mitochondrial targeting of full-length NME3. (A) Effect of PLD6 depletion on the subcellular localization of endogenous NME3. HeLa cells with GFP knock-in to the C-terminus of NME3 were transfected with PLD6 siRNA SMARTPool. (B and C) The knock-down and mitochondrial localization efficiency of endogenous NME3-GFP were quantified by Western blotting, confocal microscopy, and ImageJ analysis. (D) Effect of PLD6 depletion on the subcellular localization of exogenous NME3. NME3-GFP was transfected into control or PLD6-depleted HeLa cells. (E and F) The knock-down and mitochondrial localizing efficiency of ectopic NME3-GFP were quantified by Western blotting, confocal microscopy, and ImageJ analysis. (G and H) Subcellular distribution of NME3. HeLa cells with GFP or HA knock-in to the C-terminus of NME3 were transfected with control or PLD6 siRNA SMARTPool. After 72 h, cells were subjected to fractionation to measure the mitochondrial targeting ratio of endogenous NME3 with two different tags. Similar to their distribution observed under fluorescent microscopy, the amount of NME3-GFP or NME3-HA in mitochondrial fraction was not decreased by the depletion of PLD6. (I) Localization of ALPS-NME3-EGFP. The chimeric NME3-EGFP with its N17 replaced by the amphipathic helix lipid packing sensor (ALPS) derived from the PA-binding domain of Spo20 (62–79 amino acid) was expressed in HeLa cells and imaged together with mito-BFP. (J) The enrichment of NME3-GFP at mitochondrial contact interface prior to fusion. The dynamic distribution of NME3-GFP in two contact mitochondria was observed in Cos-7 cells co-transfected with NME3-GFP and Tom20-mCherry, which labels mitochondrial outer membrane. Arrows indicate the contact interface between two closely positioned mitochondria. Scale bars, 10 μm. Bars for inset images, 2 μm. Data are expressed as mean ± SD of three independent experiments and analyzed with Student’s t test. **P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS4.

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