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.