ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia
Luke R. G. Pike1, Kanchan Phadwal2, Anna Katharina Simon2, and Adrian L. Harris1*
1Growth
Factor Group, Cancer Research UK, Molecular Oncology Laboratories, Weatherall Institute of
Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK; 2Nuffield
Department of Medicine, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford,
UK.
*Corresponding author. Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford
University, Oxford, UK, OX3 9DS. Tel.: +44 (0)1865 222 457. Fax: +44 (0)1865 222 431. E-mail:
aharris.lab@imm.ox.ac.uk
Supplementary Information
Table 1. List of siRNA sequences used.
Gene
ATF4
Sense Sequence
CCA CGU UGG AUG ACA CUU G
Working Concentration
20nM
HRK
#1)GATCGTAGAAACACAGAAT
#2)GGGAAGCCCTTTGGAAATC
20nM (10nM each)
Scrambled Control (BNIP3
SCR)
ACGCGACACGCAGGUCGUCAU
20nM
Table 2. List of RT-qPCR primers used.
Gene
CHOP
HRK
NOXA
PUMA
Tubulinα-6
Forward Primer
AAGGCACTGAGCGTATCATGT
TACTGGCCTTGGCTGTGC
GGAGATGCCTGGGAAGAAG
GACCTCAACGCACAGTACGA
CCCCTTCAAGTTCTAGTCATGC
Reverse Primer
TGAAGATACACTTCCTTCTTGAACA
CACAGGGTTTTCACCAACCT
CCTGAGTTGAGTAGCACACTCG
GAGATTGTACAGGACCCTCCA
ATTGCCAATCTGGACACCA
Table 3. ChIP qPCR primers used.
Primer Set
HRK #1
HRK #2
HRK #3
HRK #4
Forward Primer
TCATTCTTGGAACGCTACACC
CACAGGTGCCAGACATAGAAGA
GTTCACAAAGAAAGCAGACAAGAAG
GGGTTAAAAGTTACCTCTCGGTTT
Reverse Primer
GGTTTTATACTGCCCCCTGTAATC
CCACCGCAGAGTATAACCAAG
TGGCTAGAAACCTGACCCTAAA
CTCCTTGTGTTGTGCGTTTGT
Supplementary Figure Legends
Figure S1. HRK, PUMA, and NOXA are induced by severe hypoxia. HCT116 (A-B, E-F), or MDA-MB231 (C-D) cells were exposed to severe hypoxia, after which cells were harvested at the specified
times and analyzed by RT-qPCR (A, C-F) or western blotting (B) using the indicated primers and
antibodies, respectively. ** indicates P<0.01.
Figure S2. Severe hypoxia induces HRK expression in other cell lines. HEK293 (A), HeLA (B),
MEF3T6 (C), and T47D cells (D) were exposed to severe hypoxia, after which cells were harvested
and analyzed by RT-qPCR using primers specific for HRK.
Figure S3. Chemical ER stressors induce HRK expression. (A) MCF7 cells were treated with 40M
deferoxamine mesylate (DFO), 100M cobalt (II) chloride (CoCl2), 5M arsenite, 10M MG132, 5M
MG115, 5g/mL tunicamycin, 300nM thapsigargin, 100nM bortezomib, or DMSO. After 24 hours,
cells were harvested for RT-qPCR using primers specific for HRK. (B-E) Alternatively, four cell lines
were exposed to 300nM thapsigargin and cells were harvested an analyzed by RT-qPCR using
primers specific for HRK a the designated times. ** and * indicate P<0.01 or P<0.05 by one-wayANOVA relative to the DMSO treated control, respectively.
Figure S4. HRK, PUMA, and NOXA are induced in an ATF4-dependent manner. MDA-MB-231 (A-D)
or HCT116 cells (E-H) were transfected with siRNA against ATF4 (ATF4) or a scrambled control
sequence (SCR) overnight and exposed to severe hypoxia (A) or normoxia (N) the following day.
After 48 hours, cells were harvested for analysis by western blotting or RT-qPCR using the indicated
antibodies and primers, respectively. *, **, and *** indicate P<0.05, P<0.01, and P<0.001,
respectively.
Figure S5. HRK expression in severe hypoxia is not HIF-dependent. MCF7 cells were transfected
with siRNA molecules specific for HIF1 (1), HIF2 (2), or a scrambled control (SCR), and then
exposed to normoxia, moderate hypoxia (H), or severe hypoxia (A). After 24 hours, cells were
harvested and analyzed by RT-qPCR (A) or western blotting (B) using the specified primers and
antibodies, respectively. For RT-qPCR, N=3 independent experiments were done. ** indicates P<0.01
by one-way-ANOVA for HIF2 A vs SCR A. For western blotting, representative images from at least
N=2 independent experiments are shown.
Figure S6. RNAi knockdown of HRK. MCF7 cells were transfected with a pool of sequence two
siRNA duplexes specific for HRK at a final concentration of 20nM (10nM of each). 24 hours later,
cells were exposed to severe hypoxia for a further 48 hours. Cells were then harvested and analyzed
by RT-qPCR using primers specific for HRK.
Figure S7. Knockdown of HRK reduces autophagy in HCT116 cells exposed to severe hypoxia (A) or
thapsigargin (B). HCT116 cells were transfected with siRNA molecules specific for HRK or a
scrambled control. After 24 hours, cells were exposed to normoxia (N), severe hypoxia (A) or treated
with 300nM thapsigargin (TG) for an additional 24 hours and then harvested for western blotting
using the indicated antibodies.