Cxcr4

Cytoplasmic and nuclear extracts under normoxic or hypoxic conditions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL, USA). Soluble fractions were prepared by the sampling buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1x Protease Inhibitor Cocktail, 1x Phosphatase Inhibitor Cocktail)41 (link). Ten micrograms of nuclear protein were separated on 4–8% Tris-Acetate gels (Thermo Scientific), and then transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking in 5% dry milk in Tris-buffered saline with Tween 20, the membrane was incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Detection was performed using ECL Prime (GE Healthcare, Logan, UT, USA) according to the manufacturer's instructions. The following antibodies were used: HIF-1α (NB100-449, Novus Biologicals, Littleton, CO, USA), HIF-2α (AF2997, R&D systems, NE, USA), Lamin A/C (2032S, Cell Signaling Technology, Danvers, MA, USA), goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Flt-1 (ab9540, Abcam, Camgridge, UK), CXCR4 (66042, Proteintech, Illinois, USA). The non-cropped blots of the representative images are shown in Supplementary Fig. 10. This experiment was successfully repeated three times.

The detailed procedures followed for IHC were consistent with the previous descriptions [52 (link), 53 (link)]. The information of primary antibodies is listed as follows: CTLA4 (Cell Signaling Technology, 53560S), CXCR4 (Proteintech, 60042-1-Ig), NRP1 (Proteintech, 60067-1-Ig), LGR6 (Proteintech, 17658-1-AP), and FDX1 (Proteintech, 12592-1-AP). The use and dilution ratio of primary antibodies were in accordance with the manufacturers’ instructions. Five fields were randomly selected under the 40× objective lens. The IHC scores of the samples were calculated as the sum of the scores obtained for five fields. The staining intensities and positive percentages were included in the IHC scores. The degree of staining was rated as 0, 1, 2, and 3 points, corresponding to undetected, mildly stained, moderately stained, and strongly stained, respectively. Likewise, the percentage of positivity was divided into < 5 %, from 5 % to 25 %, from 26 % to 50 %, from 51 % to 75 %, and > 75 % of positively stained cells corresponding to 0, 1, 2, 3, and 4, respectively.

The expression of proteins in Huh7 and Huh7‐R cells was detected by performing western blot (WB). Total protein from Huh7 cells was extracted applying Total Protein Extraction Kit (Cat: BC3710; Solarbio) as the protocol described. BCA Protein Assay Kit (Cat: PC0020; Solarbio) was used to assess the protein concentration. Protein samples were electrophoresed on 10% SDS/PAGE gels. Then, the separated proteins were transferred onto polyvinylidene fluoride membranes (Cat: ISEQ00010; Merck Millipore, Billerica, MA, USA). Subsequently, the membranes were incubated with the primary antibodies, CXCR4 (1 : 1000, Cat: 11073‐2‐AP; Proteintech, Wuhan, China), Cav‐1 (1 : 1000, Cat: 16447‐1‐AP; Proteintech), p‐Cav‐1 (1 : 2000, Cat: ab75876; Abcam), c‐Met (1 : 1000, Cat: 25869‐1‐AP; Proteintech), p‐c‐Met (1 : 1000, Cat: ab68141; Abcam), Raf‐1 (1 : 1000, Cat: 26863‐1‐AP; Proteintech) and EGFR (1 : 10 000, Cat: 66455‐1‐Ig; Proteintech) at 4 °C for 12 h. After blocking with 5% skim milk, the membranes were incubated with goat anti‐rabbit or goat anti‐mouse horseradish peroxidase‐conjugated secondary antibody (1 : 5000, Cat: SA00001‐2/SA00001‐1; Proteintech). GAPDH antibody (1 : 5000, Cat: 10494‐1‐AP; Proteintech) was used as a reference protein for normalization. The data were analyzed by imagej software (National Institutes of Health, Bethesda, MD, USA).