Figure S3.

Effects of optoPLDs on mitochondrial morphology. (A) Scheme of the optoPLD assay. (B and C) The increase of PLD activity induces mitochondrial clustering. optoPLD^WT or optoPLD^H170A were transfected into Cos-7 cells, and these cells were stained with mitotracker deep red prior to conversion. The optoPLDs (magenta) expressing cell was subjected to photo-conversion for 1 s per 2.5 min during the 70-min image acquisition. Boxed areas were enlarged and shown on the right panel. Scale bar, 10 μm. Bar for inset images, 2 μm. (D) The expression level of N17-GFP in control and PLD6-depleted cells. (E) Subcellular distribution of N17-GFP in PLD6-depleted cells. HeLa cells were co-transfected with N17-GFP as well as control or PLD6 siRNA SMARTPool. After 72 h, cells were subjected to fractionation to measure the mitochondrial targeting ratio of N17-GFP. Similar to their distribution observed under fluorescent microscopy, the amount of N17-GFP in mitochondrial fraction is slightly decreased by the depletion of PLD6. (F and G) Rescuing ability of PLD6-mCherry in siPLD6 cells. Wild-type PLD6-mCherry was transfected into siPLD6 HeLa cells. The rescuing abilities of these constructs were demonstrated by the significant restoration of average size of mitochondria shown in G. Scale bar, 10 μm. Bar for inset images, 2 μm. Data are expressed as mean ± SD of three independent experiments and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS3.