Supporting Information

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Supporting Information

Lee

et al.

10.1073/pnas.0812801106

Fig. S1. Inhibition of HIF-1 transcriptional activity by anthracyclines. (A) HIF-1-dependent luciferase (Luc) activity was determined in Hep3B-c1 cells treated with 0, 0.2, or 1␮M idarubicin (IDA) or epirubicin (EPI) under 20% (open bars) or 1% (filled bars) O2for 24 h. Cells were lysed and analyzed for the ratio of firefly to RenillaLuc activity. (B) HEK293 cells were exposed to 20% (open bars) or 1% (filled bars) O2for 24 h in the presence of 0, 0.2, or 1␮M IDA or EPI. Total RNA was

isolated for determination of VEGF (Left) and GLUT1 (Lower) mRNA levels by quantitative real-time reverse-transcription (qRT) PCR. The mRNA levels were normalized to the levels of 18S rRNA in each sample, and each value was expressed relative to the levels in vehicle-treated cells exposed to 20% O2. Mean⫾SEM

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Fig. S2. Mechanism of action by which anthracyclines inhibit HIF-1 activity. (A) HIF-1␣protein levels were determined by immunoblot assays of lysates prepared from HEK293 cells that were cultured in the presence of 0, 1, 5, or 10␮M daunorubicin (DNR), doxorubicin (DXR), IDA, or EPI at 1% O2for 20 h. To verify equal

loading, the blot was stripped and reprobed with a␤-actin antibody. (B) HIF-1 transactivation domain function was analyzed by using GAL4/HIF-1␣fusion proteins. HEK293 cells were cotransfected with reporter plasmid pG5-E1b-Luc, which contains five tandem GAL4-binding sites upstream of a minimal E1b gene promoter and coding sequences for firefly Luc; pSV-Renilla, which contains coding sequences forRenillaLuc downstream of a basal SV40 promoter; and an expression vector encoding GAL-O [GAL4 DNA-binding domain (DBD) only], GAL-A (GAL4-DBD/HIF-1␣531– 826), GAL-C (GAL4-DBD/HIF-1␣653– 826), or GAL-H

(GAL4-DBD/HIF-1␣786 – 826). After 24 h, transfected cells were treated with 5␮M DNR, DXR, EPI, or IDA or vehicle (Con) and exposed to 1% (filled bars) or 20%

(open bars) O2for 24 h followed by cell lysis and determination of firefly andRenillaLuc activities. The mean⫾SEM ratio of firefly toRenillaLuc activity from

three independent transfections is shown (GAL act). (C) Dimerization of HIF-1␣and HIF-1␤was determined by using GST pulldown assay. Purified GST-HIF-1␤11–510

fusion protein was incubated with lysates from HEK293 cells transfected with vector encoding FLAG-HIF-1␣in the presence or absence of 5␮M DNR, DXR, EPI, or IDA. Proteins were then pulled down with glutathione–Sepharose 4B beads and subjected to immunoblot (IB) assays by using anti-FLAG and anti-GST antibodies.

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HIF-2

IgG

Input

IP

VEGF

Promoter

PDK1

Promoter

M

Con DXR DNR Con DXR DNR

M

gDNA

20% O

₂

1% O

₂

Fig. S3. Effect of DXR and DNR on HIF-2␣DNA-binding activity. HEK293 cells were exposed to 1% or 20% O2in the presence of vehicle (Con) or 1 mM DXR

or DNR for 20 h. Input DNA was isolated from an aliquot of lysate before immunoprecipitation (IP), and lysates were then divided between anti-HIF-2␣and rabbit IgG for IP. PCR was performed by using the immunoprecipitates as template to amplifyVEGFandPDK1promoter sequences. PCR products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. M, 200-bp size marker.

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Fig. S4. Effect of DNR or DXR on HIF-1 activity in Hep3B-c1 cells. (A) HIF-1␣protein levels were determined by immunoblot assays of lysates prepared from Hep3B-c1 cells that were exposed to hypoxia (1% O2, 20 h) in the presence of vehicle control (Con) or 1, 5, or 10␮M DNR or DXR. (B) VEGF and GLUT1 mRNA

levels were determined by using qRT-PCR in Hep3B-c1 cells treated with vehicle (Con) or 0.2, 1, or 5␮M DNR or DXR under 1% (filled bars) or 20% (open bars) O2. Mean⫾SD (n⫽4) are shown.**,P⬍0.01 vs. control, Student’sttest.

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Fig. S5. Effect of DNR and DXR treatment on HIF-1 activity in cultured PC-3 cells. (A) HIF-1␣levels were determined by immunoblot assay of lysates prepared from PC-3 cells that were exposed to 1% O2for 20 h in the absence (Con) or presence of 1, 5, or 10␮M DNR or DXR. (B) Hypoxia response element (HRE)-driven

Luc activity was determined in PC-3 cells. Cells were cotransfected with HRE-driven firefly Luc and controlRenillaLuc reporter plasmids and treated with DXR or DNR for 24 h at 20% (open bars) or 1% (filled bars) O2. Cells were lysed and analyzed for the ratio of firefly toRenillaLuc activity. (C) qRT-PCR assays were

performed to quantify VEGF and GLUT1 mRNA levels in PC-3 cells treated with 0.2, 1, or 5␮M DNR or DXR at 20% (open bars) or 1% (filled bars) O2for 24 h.

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