Anti aldoa

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.

The Flag-CTL and Flag-GDPD5 plasmids were transfected into SH-SY5Y cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), as well as anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).

The GFP-CTL and GFP-HMGCS2 plasmids were transfected into OSRC2 cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), and anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).