Figure 6
Gykl1/Gk2-mediated mitochondrial clustering is PA dependent. (a) HEK293T cells transiently co-expressing HA-tagged Pld6 with GFP-tagged Gyk, Gykl1 or Gk2 were stained with an anti-HA antibody and the MitoTracker dye. Hoechst 33342 marks the nuclei. Arrows indicate areas of mitochondrial clustering. Scale bar, 10 μm. (b) Cells from a were scored for mitochondrial clustering and plotted for the percentage of cells with mitochondrial clustering. Three independent experiments were carried out with >200 cells per group. Error bars represent s.d. Statistical significance was determined by ANOVA; *P<0.05. (c) HEK293T cells were transfected with three different siRNA oligos against Pld6. At 45 h post transfection, the cells were harvested for RT-qPCR analysis. A scramble siRNA was used as a negative control. The expression level of Pld6 was calculated for each group and plotted. Error bars represent s.d. (n=3). Statistical significance was determined by ANOVA; *P<0.05. (d) HEK293T cells co-expressing GFP-PABD and HA-Gykl1 were transfected with the Pld6 siRNAs from c. At 45 h post transfection, the cells were harvested for staining using antibodies against HA and the MitoTracker dye. Hoechst 33342 marks the nuclei. Arrows indicate areas of mitochondrial clustering. Scale bar, 10 μm. (e) Cells from d were scored and plotted as described in b. Three independent experiments were carried out with >200 cells per group. Error bars represent s.d. Statistical significance was determined by ANOVA; *P<0.05. (f and g) HEK293T cells co-expressing GFP-PABD and HA-Gk2 were similarly transfected with Pld6 siRNAs, and examined (f) and analyzed (g) as described in d and e. Three independent experiments were carried out with >200 cells per group. Error bars represent s.d. Statistical significance was determined by ANOVA. *P<0.05; Scale bar: 10 μm.
