Sc 817

The technical process of immunohistochemistry (IHC) was performed as previously described^{7 (link)}. The HOXA5 staining strength was categorized into different groups depending on the positive cell percentage and positive cell staining density. The positive cell percentage was categorized into five grades: 0–3% (0), 3–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4). The positive cell staining density was also categorized into four grades: negative (0), weak brown (1), moderate brown (2), and strong brown (3). The final immunohistochemical score was calculated as follows: immunoreactivity score (IRS) = intensity score × positive score. The final IRS were categorized into three groups: negative (≤3), weak positive (>3 but ≤6), and strong positive (>6). The antibodies used were as follows: anti-HOXA5 (1:100, sc-365784, Santa Cruz); anti-Ki67 (1:100, sc-23900, Santa Cruz); anti-cyclinD1 (1:100, sc-8396, Santa Cruz); anti-p21 (1:50, sc-817, Santa Cruz); anti-β-catenin (1:50, sc-7963, Santa Cruz); anti- p53 (1:50, sc-7963, Santa Cruz). The technical process of immunocytochemistry was performed as previously described^{22 (link)}.

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).

The primary antibodies that were used were rabbit anti‐histone H4 lysine 5 acetylation (#9627, CST), rabbit anti‐lysine acetylation (ab190479; Abcam), rabbit anti‐Histone H3 lysine 18 acetylation (AJ1357a; Abgent, USA), anti‐CyclinB1 (ab151269; Abcam), rabbit anti‐histone H4 lysine 9 acetylation (07‐352; Millipore), anti‐GAPDH (92590; Millipore, USA), anti‐CTGF (ab6992; Abcam, USA), mouse anti‐p21 (SC‐817; Santa Cruz), rabbit anti‐TXNIP (ab188865, Abcam,USA), goat anti‐gamma H2AX (Santa Cruz), mouse anti‐BST2 (ab88523; Abcam), rabbit anti‐Histone H3 lysine 4 trimethylation (07‐437; Millipore), rabbit anti‐Histone H3 trimethylation lysine 27 (07‐449; Millipore), rabbit anti‐Histone H3 trimethylation lysine 9 (07‐442; Millipore), and goat anti‐GFP (ab6673; Abcam). The secondary antibodies that were used were anti‐rabbit IgG‐HRP (ab191866 Abcam), anti‐mouse IgG‐HRP (ab193651 and ab193652; Abcam), donkey anti‐goat‐HRP (31400, Pierce), and DyLight 488 Goat Anti‐Rabbit (111‐055‐003, Jackson).