Download Supplemental Figure Legends Figure S1. Normal beta

Supplemental Figure Legends
Figure S1. Normal beta-islets in Wwox-deficient mice
(A) Pancreas from Wwox wild-type (WT) and KO mice were stained for beta
cells marker Pdx4 (red) and insulin (green) to distinguish -islets.
Figure S2. WWOX deficiency is associated with enhanced glycolysis
(A) Lactate levels in the serum of Wwox WT and KO mice (WT=4, KO=12).
Error bars indicate ±SEM.
(B) NADPH levels were measured in the livers of the Wwox WT and KO mice
(WT=3, KO=3). Error bars indicate ±SEM.
Figure S3. WWOX directly inhibits expression of glycolytic genes
(A)
Expression of glycolytic genes in WWOX KO MEFs. RNA was purified
from WWOX WT and KO MEFs cells (WT=3, KO=3) and real-time PCR
(RT-PCR) was performed using Glucose Metabolism PCR Array
(SAbiosciences), keeping a threshold of 0.4 as confidence value in the
threshold cycle (Ct).
(B)
Expression of glycolytic genes in WWOX KO MEFs that are transcriped
by HIF1α (labled in red).
(C)
Expression of Tricarboxylic Acid Cycle (TCA) genes in KO MEFs. RNA
was purified from WWOX WT and KO MEFs cells (WT=3, KO=3) and realtime PCR (RT-PCR) was performed using Glucose Metabolism PCR Array
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(SAbiosciences), keeping a threshold of 0.4 as confidence value in the
threshold cycle (Ct).
(D) Expression of pentose phosphate pathway (PPP) genes in WWOX KO
MEFs. RNA was purified from WWOX WT and KO MEFs cells (WT=3,
KO=3) and real-time PCR (RT-PCR) was performed using Glucose
Metabolism PCR Array (SAbiosciences), keeping a threshold of 0.4 as
confidence value in the threshold cycle (Ct).
(E) Expression of gluconeogenesis pathway genes in WWOX KO MEFs. RNA
was purified from WWOX WT and KO MEFs cells (WT=3, KO=3) and realtime PCR (RT-PCR) was performed using Glucose Metabolism PCR Array
(SAbiosciences), keeping a threshold of 0.4 as confidence value in the
threshold cycle (Ct).
(F) Expression of gluconeogenesis pathway genes in WWOX KO liver. RNA
was purified from the liver of Wwox WT and KO mice (WT=3, KO=3) and
real-time PCR (RT-PCR) was performed using Glucose Metabolism PCR
Array (SAbiosciences), keeping a threshold of 0.4 as confidence value in
the threshold cycle (Ct).
Figure S4. WWOX Directly Inhibits HIF1α trans-activation function under
normoxic and hypoxic conditions.
A) Expression of HIF1α target genes in glycolysis in MCF7 par and MCF7
SH WWOX using real time PCR. * is P-value<0.05. ** is P-value<0.01.
Error bars indicate ±STDV.
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(A) Expression of HIF1α target genes in glycolysis in MCF7 SH WWOX and
MCF7 SH WWOX (+WWOX) using real time PCR. * is P-value<0.05. ** is
P-value<0.01. Error bars indicate ±STDV.
(B) A luciferase reporter gene under the regulation of Hypoxia-Responsive
Elements (HRE) was co-transfected with empty vector, WWOX, HA-HIF1α
with empty vector, and HA-HIF1α with WWOX plasmids into 293T cells
and subjected to normoxia (20% O2) (green) or Hypoxia (1% O2) (blue)
conditions for 6 hr. Extracts were analyzed for Luciferase activity. Error
bars indicate ±SEM.
(C)
mRNA expression of HIF1α gene in the skeletal muscles of Wwox KO
and WT mice that were infected by empty vector or WWOX, using real
time PCR. Error bars indicate ±SEM.
Figure S5. Knocking down HIF1α Completely Rescues the Metabolic
Phenotype in vitro and in vivo, and WWOX interacts with HIF1α by its
first WW domain
(A) WWOX KO MEFs were infected with a WWOX or WWOX-WFPA or Sh
HIF1α expressing lentivirus and WWOX WT MEFs were infected with a
WWOX or WWOX-WFPA or Sh HIF1α expressing lentivirus. Infected cells
were selected by puromycin, and following expansion, cells were assayed
for glucose uptake following 1 hr incubation with 2-NBDG. Error bars
indicate ±SEM. (EV=3, WWOX=3, WFPA=3, Sh HIF1α=3)
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(B) GLUT1 and PHD3 (HIF1α target genes) expression in the skeletal muscles
of Wwox KO digoxin treated mice compared to placebo treated KO mice.
Error bars indicate ±STDV. (KO with digoxin=2, KO with placebo=2)
(C) GLUT1 and PHD3 (HIF1α target genes) expression in the skeletal muscles
of Wwox WT digoxin treated mice compared to placebo treated WT mice.
Error bars indicate ±STDV. (WT with digoxin=2, WT with placebo=2)
Figure S6. WWOX interacts physically with HIF1α through its first WW
domain.
(A)
WWOX interacts with Hif1a by its first WW domain, 293t cells were
transficted by several WWOX domains that fused with glutathione Stransferase, GST WW1 or GST WW2 or GST WW1+2 or GST SDR,
hypoxia (1%O2) for 6 h or 1ug of MG132 was added to stabilize Hif1a.
(B) Endogenous WWOX physically interacts with endogenous HIF1α.
HEK293T cells were treated with MG132 (1μM) for 4 hours or by hypoxia
for 6 hours to stabilize HIF1α followed by immunoprecipitattion (IP) with
anti-WWOX or anti-HIF1α antibody. Immunoblotting was done using antiWWOX, anti-HIF1a or anti-HIF1α-OH antibodies. Anti-IgG was used as a
negtaive control for IP.
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