Hif 1α

The chromatin immunoprecipitation assay was performed using the Chromatin Immunoprecipitation Assay kit (EZ ChIP; Millipore) according to the manufacturer’s instructions. Briefly, HEK293 cells were transfected with pCDNA3.1-HIF-1A or human C/EBPδ. C/EBPδ plasmid construct was engineered to express the complete open reading frame with an expression Flag tag. Forty-eight hours after transfection, the cells were then cross-linked by 1% formaldehyde for 10 minutes. The formaldehyde was quenched using 2 M glycine for 5 minutes at room temperature before harvest. Cells were collected by centrifugation in phosphate-buffered saline containing protease inhibitors and were lysed in SDS-lysis buffer. Soluble chromatin was prepared after sonication to an average DNA length of 200 to 500 base pairs. Fragmented chromatin was immunoprecipitated using antibodies against HIF-1α (Millipore) or Flag together with Protein A/G PLUS-Agarose overnight at 4°C on a rotating platform. The agarose beads were washed, chromatin extracted and protein–DNA cross-links reversed. DNA was purified and was analyzed by PCR using the specific primers. Normal immunoglobulin G was used as a negative control. Anti-RNA Polymerase II was used as positive control. The total input was the supernatant from the no-antibody control.

Total cell lysates or nuclear extracts were prepared and proteins were separated using SDS-PAGE (10%). Proteins in the gel were transferred electrophoretically to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked in 5% milk and immunoblotted with antibodies against the following proteins, including HIF-1α (Millipore), ARNT (HIF-1β) (Abcam Inc., Cambridge, MA, USA), AHR (GeneTex, Irvine, CA, USA), TP53 (Dako, Copenhagen, Denmark), CDKN1A (p21) (GeneTex), CDK4 (GeneTex), p-RB1 (GeneTex), GAPDH (GeneTex), NDRG1 (Abcam), TUBA1A (α-tubulin) (Novus Biologicals, Littleton, CO, USA) and LMNA (Lamin A/B) (Novus). Bound primary antibodies were reacted with horseradish peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG (GeneTex) and detected with the chemiluminescent western blotting system (Millipore).

Cells were harvested by washing with cold PBS and lysed in cell lysis buffer (150 mM NaCl, 0.5% NP-40, 50 mM Tris-HCL, 10% glycerol, 1% SDS, pH 7.5, supplemented with 1x protease inhibitor cocktail (Roche)) on ice. The extracted proteins were separated in homemade SDS-PAGE gel and transferred onto PVDF membrane. Blocking was done with 5% skim milk (BD) in TBST (TBS+0.1% Tween-20). After blocking, the membrane was incubated with primary antibody overnight at 4°C and washed with TBST for 3 times, then incubated with HRP-linked secondary antibody (Cell Signaling Technology, #7074 and #7076) for 1 hour and washed with TBST. The signal was developed with Luminata Crescendo Western HRP Substrate (WBLUR0500, Millipore) and captured on X-ray film. Primary antibodies used in the current study included Anti-HA (660002, Biolegend), HIF1α (SAB2702132, Millipore), α-Tubulin (T6199, Sigma). Protein bands were quantified using ImageJ.

Antibodies used in this study were as follows: BATF2 (ab157466, Abcam, London, UK); BATF2 (Thermo Fisher, PA5-43094); BATF2 (241887, Santa Cruz Biotechnology, CA, USA); CD63 (Abcam, ab217345); CD9 (Abcam, ab92726); TSG101 (Abcam, ab125011); SDF-1α (Abcam, ab9797); SDF-1α (Abcam, ab181018); HIF-1α (H6536, Sigma); CXCR4 (Abcam, ab135170); VEGF (ab46154, Abcam); CD33 (Abcam, ab203253); CD14 (Abcam, ab203294); Gr-1 (108448, Biolegend); CD11b (Abcam, ab13357); CXCR7 (Biolegend, #391405); SV40 Large T Antigen (CST, #15729, CA, USA); MMP-9 (CST, #13667); 10 nm gold-conjugated goat anti-rabbit secondary antibody (bs-0437P-Gold, Bioss, China), GAPDH (sc365062, Santa Cruz Biotechnology), and β-Actin (sc-47778, Santa Cruz Biotechnology).

Whole cell lysates were prepared by RIPA kits (Atto, Tokyo, Japan) while the protein concentration was accomplished by a SMART^TM micro BCA kit (Intron biotechnology). Proteins were separated on Bis-Tris pre-cast gels and transferred to PVDF membrane (Bio Rad, CA, USA). Primary antibodies against PARP (BD), caspase-3, CD133, VEGF, Chk1, p-Chk1, Chk2, p-Chk2, PCNA, MCM2, p-ATM (Abcam, Cambridge, UK), mTOR, p-mTOR, AMPKα, p-AMPKα (Cell signaling, MA, USA), HIF-1α (Sigma-Aldrich), β-actin, and β-tubulin (Santa Cruz, CA, USA) were used at 1:1000. Peroxidase conjugated secondary antibody were against rabbit or mouse (Jackson Immunoreasearch, MD, USA) diluted 1:5000. Chemiluminescent solution was obtained from Intron biotechnology and detected using Fuji RX film.

The following RNAi were employed, siMCT4 (Santa Cruz, SC-45892), HIF1α(Sigma, NM_181054). The shRNA against MCT4 was introduced by lenti-viral transduction as previously published [18 (link)].