Anti eno1

The IHC staining was used to examine the expression levels of c-Myc and glycolytic enzymes. The paraffin-embedded sections were incubated in a pressure cooker containing antigen retrieval buffer (pH 6.0) (Solarbio; G1202). Next, the slides were uniformly covered with 3% bovine serum albumin (Solarbio; A8020), and probed with the following primary antibodies prepared in a wet box at 4 °C overnight: anti-c-Myc (Servicebio; GB13076), anti-GLUT1 (Proteintech, 21,829–1-AP), anti-ENO1 (Proteintech, 11,204–1-AP), anti-PGK1 (Proteintech, 17,811–1-AP), anti-ALDOA (Proteintech, 11,217–1-AP), anti-HK1 (Proteintech, 19,662–1-AP), and anti-LDHA (Proteintech, 19,987–1-AP) antibodies. Next, the horseradish peroxidase (HRP)-labeled secondary antibody was used for 50 min. Then, the sections were then counterstained with hematoxylin.
The stained sections were scanned using Pannoramic MIDI and analyzed using Quant Center, which automatically identified all the strongly positive, moderately positive, weakly positive, and negative areas in the tissue sections. The H-scores were calculated as ∑ (percentage of intensity × intensity). The staining intensity was divided into three categories—strong, moderate, and weak—and the corresponding score was 3, 2, and 1, respectively.

IHC was used to measure protein expression of the specimens of participants and animals with the specific primary antibody at 4℃ overnight in a humidified chamber. The primary antibody included anti-KMT5A (ProteinTech, Wuhan, China), anti-ENO1 (Proteintech, Wuhan, China), anti-regulatory factor X1 (RFX1, Santa Cruz Biotechnology, Santa Cruz, CA), anti-vimentin (Proteintech, Wuhan, China), anti-CD31 (Proteintech, Wuhan, China) and anti-αSMA (Cell Signaling Technology, Danvers, MA) antibodies.

Tumor tissues and cells were washed with PBS and underwent lysis with RIPA buffer (Pierce) supplemented with protease inhibitors (Boster Biological Technology, China). Protein quantitation utilized the BCA assay kit (Beyotime Biotechnology). Equal amounts of total protein were resolved by SDS-PAGE and underwent transfer onto polyvinylidene fluoride membranes (Millipore, USA). Western blot membranes underwent overnight incubation at 4 °C with anti-FAM126A (1:1000; Sigma, #84668), anti-ENO1 (1:1000; Proteintech, #11204), anti-GHPDH (1:1000; Proteintech, #60004), anti‑E‑cadherin (1:1000; Proteintech, #20874), anti-N-cadherin (1:1000; Proteintech, #22018), anti‑vimentin (1:1,000; Proteintech, #10366), anti-snail (1:1000; Proteintech, #13099), anti-cyclin D1 (1:1000; Cell Signaling Technology [CST], #55506), anti-cyclin E1 (1:1000; CST, #4136), anti-CDK 2 (1:1000; CST, #2561), anti-CDK4 (1:1,000; CST, #12790), anti-AKT (1:1000; CST, #4691), anti-p-AKT (1:2000; CST, #4060), anti-PI3K (1:1000; CST, #4249), anti-p-PI3K (1:1000; CST, #17366), anti-Ki-67(1:800; CST, #9449) and anti-PCNA (1:1000; CST, #2561) primary antibodies. This was followed by incubation with HRP-linked anti-rabbit IgG (Boster, #BA1041). Enhanced chemiluminescence reagent (Proteintech, #7003) was used to detect immunoreactive signals.