Supplementary Table and Figure Legends
Supplementary Table 1. List of genes significantly regulated following FOXO3a
activation in DL23 cells. DL23 cells were treated with ethanol or 100 nM 4-OHT for
24 hours. Total RNA was subjected to gene expression analysis (Affymetrix human
Exon Array). Signal estimates were generated using RMA. FOXO3a regulated genes
were selected using a false discovery rate of 0.05. Annotations were derived from
Affymetrix release HuEx-1_0-st-v2.na24.hg18.
Supplementary Figure S1. Activation of endogenous FOXO by inhibition of PI3kinase leads to downregulation of mitochondrial genes. Parental DLD-1 cells were
treated with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitors LY-294002 (20
µM) and PI-103 (0.5 µM) for 24 h. Expression levels of PGC1, PRC, TFAM, SOD2
and Mxi1 were determined by qRT-PCR. Data representative of 2 independent
experiments are shown. Data are mean ± SEM.
Supplementary Figure S2. FOXO3a does not regulate the activity of a TFAM
reporter construct. (a) Schematic representation of mtTFA-luc WT. This reporter
construct carries proximal sequences from the promoter of the human TFAM gene
containing NRF, NRF2 and SP1 binding sites (mtTFA-luc WT). (b) DL23 cells were
transfected with different amounts of mtTFA-luc WT and treated with solvent or 4OHT for 24 hours. Firefly luciferase activity was normalised to the activity of a
cotransfected Renilla-luciferase expression construct. Data are mean ± SEM, n=4.
Supplementary Figure S3. c-Myc RNA levels in DL23 and DL23+c-Myc cells.
DL23 cells were infected with pWZL-Blast-c-Myc and selected for 96 hours. Cells
were then treated with 4-OHT or solvent for 24 h. Expression levels of c-Myc
following FOXO3a activation in parental cells (white and grey bars) or c-Myc
expressing cells (hatched and black bars) was determined by qRT-PCR. Data are
mean ± SEM, n=3.
Supplementary Figure S4. FOXO3a does not affect total Mitotracker green
fluorescence levels. RPE-F cells were treated with ethanol or 4-OHT for 48 h, or with
DMSO or 10 µM Resveratrol for 48 hours. Cells were fixed in formaldehyde, stained
using 100 nM mitotracker green (MTG) and fluorescence was measured by flow
cytometry. Data are mean ± SEM. * indicates statistical significance as determined by
Student’s t-test (P < 0.05, n=3). N.S. = non signicficant.
Supplementary Figure S5. Effect of inhibition of mitochondrial fission on oxygen
consumption in RPE-F cells. (a) FOXO3a does not upregulate mitochondrial fission
regulators FIS1 and DRP1. RPE-F cells were treated with ethanol or 4-OHT for 48 h.
Expression levels of FIS1 and DRP1 were determined by qRT-PCR. (b) The DRP1
inhibitor Mdivi-1 does not block the downregulation of oxygen consumption rate
induced by FOXO3a activation. RPE-F cells were treated with ethanol or 4-OHT for
48h and 10 µM Mdivi-1 or solvent were added for the final 24 h. Oxygen
consumption rate (OCR) was measured using Oxygen Biosensor Plates in the
presence or absence of 2 µM FCCP. Data are mean ± SEM, n=3.
Supplementary Figure S6. Effect of FOXO3a activation on HIF-1mRNA levels
in hypoxia. RPE-F and DL23 cells were treated with ethanol or 4-OHT for a total of
48 h and placed in normoxia (20% O2) or hypoxia (<0.5% O2) for the final 24 h.
mRNA levels of HIF-1 were determined by qRT-PCR. Data are mean ± SEM, n=3.
Supplementary Figure S7. Effect of PHD inhibition and VHL silencing on HIF1expression in RPE-F cells. (a) RPE-F cells were treated with ethanol or 4-OHT
for a total of 48 h and placed in normoxia (20% O2) or hypoxia (<0.5% O2) for the
final 24 h or treated with 1 mM DMOG for the final 4 h. Expression of HIF-1 was
determined by immunoblotting. -actin is shown as a loading control. (b) RPE-F cells
were transfected with 100 nM of control or VHL siRNA for 72 h and treated with
ethanol or solvent during the final 24 h. Expression of HIF-1 was determined by
immunoblotting. -actin is shown as a loading control. (c) Silencing of VHL was
confirmed in RPE-F cells transfected in parallel by qRT-PCR.