Ab27595

Western blotting was performed as previously described^{22 (link), 23 (link), 46 (link)}. The lysates from the xenografts or cell lines with different treatments were homogenized in RIPA lysis buffer using a homogenizer. The supernatants were collected by centrifugation (12,000 rpm at 4 °C for 25 min; Beckman GS-6R). Protein was resolved by electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were probed with monoclonal HIF-1α (NB100–105, Novus biological, Littleton, CO, USA), Bax (2772 S, Cell signaling, Boston, MA, USA), Bcl-2 (3498S, Cell signaling), caspase-3 (9661S, Cell signaling), PARP (5625, Cell signaling), and PCNA (18197, Abcam, Cambridge, MA, USA), as well as p21 (Sc817, Santa Cruz, CA, USA), CDK4 (Sc70831, Santa Cruz), SP1 (ab27595, Abcam), HK2 (2772S, Cell signaling), PKM2 (4053S, Cell signaling), LDH-A (3582S, Cell signaling), and PDK1 (3820 S, Cell signaling). Following incubation with the primary antibody overnight at 4 °C, membranes were washed with Tris-buffered saline (pH 7.2) containing 0.05% Tween-20 and subsequently incubated with horse radish peroxidase (HRP)-conjugated anti-mouse (Sc-2005, Santa Cruz) or anti-rabbit (Sc-2357, Santa Cruz) secondary antibody for 1 h at room temperature. Bands of interest were analyzed using the ChemiDocTM Touch Imaging System (BIO-RAD, Hercules, CA, USA).

To maintain the 3D structure of the primary culture tumor cells, BME-cells mixture was aspirated from the 24-well plates gently and completely, then it was embedded in HistoGel (Biocoat, Corning, NY, USA). All samples were fixed in 4% paraformaldehyde before embedded in paraffin. The slides were incubated overnight with primary antibody Rabbit anti-ER antibody (1:5, ab27595), anti-PR (1:150, ab63605), anti-HER2 (1:600, ab134182), and anti-Ki67 (1:150, ab16667) which were purchased from Abcam (Cambridge, MA, UK). The DAB kit was purchased from Zhongshan Goldenbridge Biotechnology Company (Beijing, China). All procedures were carried out according to the manufacturer’s instructions.

Whole-mount estrogen receptor (ESR) staining was performed according to the protocol for whole-mount PECAM staining^{55 (link)}. Briefly, embryos were collected in PBS on ice and fixed in 4% PFA in PBS overnight at 4 °C. After washing three times in PBS, embryos were dehydrated through a methanol gradient (25, 50, 75, and 100% methanol in PBS) for 15 min each at room temperature. Embryos were bleached in 5% hydrogen peroxide in pure methanol for 2 h at 4 °C to inhibit endogenous peroxidase activity. Then the embryos were rehydrated through 100, 75, 50, and 25% methanol in PBS for 10 min each at room temperature. For embryos with tails covering part of heart surface, tails were removed before staining. Embryos were blocked in PBS-containing 0.1% Triton X-100 and 5% normal donkey serum for 1 h at 4 °C. Samples were then incubated in block solution containing 50% ESR antibody (Abcam, ab27595) overnight at 4 °C, followed by five times wash in PBS-containing 5% normal donkey serum. Samples were then incubated with secondary antibody anti-rabbit conjugated with peroxidase (Vector lab, 1:500) in blocking solution for 10 h, followed by five times PBS-containing 5% normal donkey serum wash. Finally, embryos were developed by chromogen DAB (Vector Lab) at room temperature to the desired extent. Images were taken under Leica stereomicroscope (M165 FC).