Legends to Figures
Figure 1. eEF2 is detected in the mitochondria. a. The mitochondria and cytosolic fractions of MCF7, MDA-MB-231, LN229, or SVGp12 cells were separated and eEF2, p-eEF2 and eEF2 kinase were examined by western blot. COX IV was used as a marker of mitochondria fraction. b. MCF7 cells were incubated with Mitotracker Red® and eEF2 antibody, followed by incubation with a second antibody and AlexaFluor®488 Green. Green: eEF2; red: mitochondria .
Figure 2. Effect of eEF2 expression on the length of mitochondria. MCF7 cells (a) or MEFs (b) were transfected with sieEF2 or non-targeting control RNA for 72 hours, and then were stained with Mitotracker Red® and observed under a confocal microscope. The lengths of mitochondria were measured using the NIS-elements AR Analysis software (Nikon); five independent images were used for measuring the mitochondria length (n=5).c. MCF7 cells with or without silencing of eEF2 expression were harvested by trypsinization, then were fixed and embedded in spur resin. Ninety nm thin sections were cut and examined at 80 Kv under a JEOL 1200 EX transmission electron microscope. The bar: 500 nm scale .d. MCF7 cells were transfected with pcDNA eEF2 expression vector or control pcDNA 3.1 His6C vector for 72 hours, and then were stained with Mitotracker Red® and observed under a confocal microscope. The lengths of mitochondria were measured using the NIS-elements AR Analysis software (Nikon); five independent images were used for measuring the mitochondria length (n=5).
Figure 3. Knockdown of eEF2 alters mitochondrial metabolism.a. MCF7 cells were transfected with 100 or 200nM of sieEF2 or non-targeting control RNA for 72 hours. The transfected cells were then seeded in 96-well plates (4 X 104/well). OCR were time-serially analyzed using an XFe96 analyzer. b. MDA-MB-231 or MCF7 cells were transfected with 100 or 200nM of sieEF2 or non-targeting control RNA for 72 hours, followed by western blot analysis of eEF2 and SOD2. GAPDH is used as a loading control.
Figure 4. eEF2 binds to and colocalizes with Drp1 on the mitochondria. a. HEK293 cells were transfected with sieEF2 or non-targeting control RNA for 72 hours. The cytosol and mitochondria fractions were prepared for western blot analysis. b. HEK293 cells were transfected with pcDNA 3.1 His6C or pcDNA eEF2 for 72 hours. The cytosol and mitochondria fractions were prepared for western blot analysis. PHB1 (prohibitin 1) was used as a mitochondrial marker.c. HEK 293 cells were transfected with pcDNA eEF2 or eEF2 GTP mutant (M) and pGW1 containing Drp1 for 72 hours. Left upper panel: The agarose-conjugated Drp1 antibody was incubated with the lysates of the cells transfected with the pcDNA 3.1 His containing eEF2 or eEF2 GTP mutant for 24 hours. The eluted protein complex was examined by western blot. Right upper panel: The agarose-conjugated eEF2 antibody was incubated with the lysates of the cells overexpressing Drp1 for 24 hours. The eluted protein complex was examined by Western blot. Rabbit IgG was used as a control. d. GST-eEF2, GST- eEF2 GTP mutant, and GST-Drp1 proteins were precipitated with magnetic GST beads, and then incubated with the lysates of the cells transfected with either eEF2, eEF2 GTP mutant or Drp1. The eluted proteins were analyzed by Western blot. e. MCF7 cells were seeded in coverslip and incubated with eEF2 and Drp1 antibodies, followed by incubation with the second antibodies, Alexa Fluor®488 Green and AX568 Fluor®Red.
Figure 5. Validation of the effect of eEF2 on mitochondria fission . a. Effect of the eEF2 mutant on mitochondrial fission. HEK293 cells were transfected with a control vector (pcDNA 3.1 His6C), expression vectors of eEF2 or eEF2 GTP deletion mutant for 72 hours. The transfected cells were stained with Mitotracker Red® and observed under a confocal microscope. The lengths of mitochondria were measured using NIS-elements AR Analysis software (Nikon); five independent images were used (n=5). b.Rescue of the eEF-2 silencing effect on mitochondrial fission by transfection of cells with a non-degradable eEF-2 cDNA. MCF cells harboring an empty vector, a wild-type or non-degradable eEF-2 expression vector (2 µg) were transfected with an eEF-2-targeted siRNA (200 nM) for 72 hours. Mitochondrial lengths were measure as described above.
Fig 6. Effect of eEF2 on GTPase activity of Drp1. a. Based on a functional analysis of eEF2 protein using UniProt, the GTP binding motif of eEF2 was deleted using mutagenesis PCR approach. b. eEF2, eEF2 deletion mutant (eEF2 mu) and Drp1 proteins expressed in E. Coli cells were purified using magnetic GST bead under phosphate and nucleotide free condition. Proteins of Drp1 and eEF2 or Drp1 and eEF2 GTP deletion mutant were mixed (vol:vol: 1 : 1= 2 μg : 2 μg). GTPase activity was measured using the colorimetric GTPase activity assay kit from Sigma.