Legends to Supplemental Figures
S.Fig.1. Presence of eEF2 on the mitochondria of LN229 cells and its effect on mitochondrial fission. a. LN229 cells were transfected with 200nM of sieEF2 or non-targeting control RNA for 72 hours, and then were stained with Mitotracker Red® and incubated with eEF2 antibody. The cells were observed under a confocal microscope and the lengths of mitochondria were measured using the NIS-elements AR Analysis software (Nikon), and five independent images were used for measuring the mitochondria length (n=5).
S.Fig. 2. Real-time live cell imaging. Cells with or without silencing of eEF2 expression were grown in the glass bottom dishes and stained with 100 nM of Mitotracker green for 45 minutes. Then, the cells were washed with pre-warmed culture media three times before imaging. Fluorescence images were captured using a BioRad 2100 Radiance laser scanning confocal microscope.
S. Fig.3. Effect of eEF2 on mitochondrial fission under hypoxic condition . MCF-7 cells with or without depletion of eEF2 were subjected to hypoxia or normoxia for 24 hrs, 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) and five independent images were used for measuring the mitochondria length (n=5).
S. Fig. 4. Effect of eEF2 kinase on mitochondria fission. a.Mouse melanoma B16 OVA cells with or without knockout of eEF2 kinase were analyzed for their expressions of eEF2 kinase, eEF2 and phospho-eEF2 by Western blot. b. The cells were stained with Mitotracker green® and observed under a confocal microscope, and the length of mitochondria were measured using NIS-elements AR Analysis software (Nikon); five independent images were used for measurement of the mitochondria length (n=5).
S. Fig. 5. Effect of eEF2 on cell proliferation. a. MCF7 cells were transfected with sieEF2 or non-targeting control RNA (200 nM) for 72 hrs, and then were seeded in 6-well plates (3 X105cell/well). After 3 days, cells were counted using a hemotocytometer.b. MCF7 cells were transfected with pcDNA, pcDNA eEF2 or pcDNA eEF2 mutant for 72 hrs, and then were seeded in 6-well plates (3 X105 cells/ well). After 3 days, cells were counted using a hemotocytometer. The data shown are mean ± S.D. of a representative from three identical experiments.