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.