Cell culture

Colorectal cancer cells (HCT116, RKO, HCT8, HT29) and normal epithelia cell line (NCM460) were obtained from the Cell Bank, Chinese Academy of Sciences. Cells were cultured in DMEM. Fetal bovine serum (FBS, 10%) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) were added to all media. All cells were cultured in a constant temperature incubator (5% CO2, 37 °C). Cell transfection was performed using Lipofectamine 8000 according to the instructions.

Immunohistochemistry

After dewaxing and rehydration of tissue sections, they were placed in EDTA solution for high-temperature retrieval for 30 min. After natural cooling to room temperature, endogenous peroxidase inhibitor was used to block endogenous peroxidase for 15 min. The tissue sections were washed with PBS for 1–2 times, and then incubated with Sox9 antibody (CST, #34330, 1:500) and Ki67 antibody (proteintech, 27309-1-AP, 1:8000) at 4 °C overnight. Then, the tissues were washed with PBS, and incubated with secondary antibody at room temperature for 1 h. 3,3ʹ-diaminobenzidine (DAB) was used to develop the immunohistochemical signals. All tissue sections were counterstained with hematoxylin, and the staining intensity and protein expression level were automatically scored by the Vectra2 system.

HE staining

After dewaxing and rehydration of tissue sections, the slices were put into hematoxylin staining solution for 3 min. After rinsing with water and differentiation with 1% hydrochloric acid alcohol for a few seconds, the tissues were then counterstained with 0.6% ammonia water for bluing. Finally, the tissues were put into eosin staining solution for 1–3 min. The slices were dried, dehydrated, and then mounted for photography under a microscope.

The mouse model induced by AOM/DSS

Pathogen-free 12-week-old male KDM6Af/f mice or Villin-Cre; KDM6A f/f mice on a BALB/c genetic background were housed under SPF conditions with free access to food and water during the experiments. Mice were injected intraperitoneally with AOM (12 mg/kg body weight) dissolved in physiological saline. After a five-day interval, the mice were then administered 2% DSS through their drinking water for a duration of five days. Following this, regular water was provided for a period of 16 days. This cycle was repeated twice to ensure consistent exposure to AOM and DSS. Animals were killed at the indicated time point for histological analysis. All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals set by the Committee for Animal Experimentation of Hainan Medical University, and the protocols were approved by the committee.

Western blot

Cells were washed twice with PBS and then lysed on ice using RIPA lysis buffer containing protease and phosphatase inhibitors. The cell lysate was centrifuged and the supernatant was collected. The protein concentration was quantified using the BCA protein assay kit. Equal amounts of protein were loaded onto an SDS-PAGE gel for electrophoresis. After protein separation, the proteins were transferred onto a PVDF membrane. The membrane was subsequently incubated with specific primary antibodies overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies for 1–2 h. The immunosignals were detected using a chemiluminescent substrate (Millipore, WBKLS0050) and analyzed using Image Lab software. The primary antibodies used in this experiment included: KDM6A (proteintech, 23984-1-AP, 1:1000), tubulin (Santa Cruz Biotechnolog, sc-5286, 1:4000), LDHA (proteintech, 19987-1-AP, 1:1000), Flag (proteintech, 66008-4-lG, 1:1000), and HIF-1α (Cell signaling technology, #36169, 1:1000) (Additional file 1).

CCK8 assay

Cells were seeded at a density of 1 × 103 cells per well in a 96-well plate and incubated in a constant temperature incubator set at 37 °C with 5% CO2. The next day, the old medium was replaced with fresh medium containing 10% CCK8, and the plate was incubated in the incubator for 2 h. The absorbance at 450 nm was measured daily starting from the initiation of the experiment.

Colony formation assay for the soft agar

Cells were seeded in a 24-well plate, and the following day, the cell confluence reached approximately 50%. Subsequently, the cells were digested and a cell suspension was prepared. The lower agar layer was prepared according to the following formula: 20% FBS, 40% 2 × RPMI1640 (Basal Medium Eagle), 40% agar (1.25% w/v). 400 μl of the mixture was added to each well, and the plate was then placed in a 37 °C incubator until the gel solidified. The upper agar layer was prepared and mixed evenly with the cell suspension. 400 μl (1 × 103 cells) was added to each well and placed in a constant temperature incubator (37 °C, 5% CO2) for 10–14 days. The formula for the upper agar layer was: 25% FBS, 37.5% 2 × RPMI1640, 37.5% agar (1.0% w/v), 0.8% 2 mM l-glutamine. Five random fields were selected under a microscope for clone counting.

EdU assay

Cells were seeded in a 96-well plate at a density of 2 × 104 cells per well. The Cell-Light EdU Apollo567 In Vitro Kit (Ruibo Biotechnology, C10310-1) was used for detection. Images were captured and analyzed using a high-resolution fluorescence microscope.

Measurement of the lactate content

Lactate production in the medium was detected by using Enzychrom l-Lactate Assay Kit. Results were normalized to the total protein concentration.

Knockdown of KDM6A and LDHA

shRNA targeting KDM6A and LDHA was designed using the Sigma website, and clone them into the pLKO.1-puro vector. The lentivirus was packaged using 293T cells, with packaging vectors psPAX2 and pMD2.G. Then, the viral supernatant was collected, concentrates using PEG8000 at 4 °C and 1600g for 1 h, removed the supernatant, and dissolved in 2 ml DMEM. Cells with 50%–60% confluency in a 6-well plate were carefully selected for the experiment. To introduce the lentivirus, 400 µl of the lentivirus was added to the cells and incubated overnight. After two days, the cells were further treated with puromycin (1 mg/ml) for a duration of 4 days to facilitate the selection of stable cell lines. To assess the expression levels of KDM6A and LDHA, Western blot analysis was performed. This technique allowed for the detection and quantification of the proteins of interest in the selected cell lines.

Immunoprecipitation

Immunoprecipitation (IP) is used to detect the interaction between endogenously expressed proteins. After treating the cells, they are lysed using an IP lysis buffer containing proteinase and phosphatase inhibitors. After centrifugation, the lysate is separated and the supernatant is carefully collected. Subsequently, 1 μg of antibody is introduced and left to incubate with the supernatant overnight at a temperature of 4 °C. On the following day, 40 μl of Protein A/G beads (bimake.com, B23202) are added and allowed to incubate for 4 h at 4 °C. The beads are then washed three times with wash buffer before 1× loading buffer is added for subsequent Western blotting analysis.

ChIP

Cells were seeded in a 10-cm dish. When the confluency reached 90%, the cells were cross-linked with 1% formaldehyde for 10 min and incubated with 125 mM glycine at room temperature for 3 min. The cells underwent two washes with pre-chilled PBS. Subsequently, the cells were gathered in 2 ml of DTT solution (100 mM pH 9.5 Tris–HCl, 10 mM DTT) and left to incubate at room temperature for 10 min. Afterward, the cells were centrifuged at 4 °C and 5000g for 5 min. The resulting cell pellet was then suspended in 150 μl of SDS lysis buffer (50 mM Tris–HCl at pH 8.0, 2 mM EDTA, and 1% SDS) containing protease and phosphatase inhibitors. DNA fragments were sheared to an average size of approximately 500 bp using a 12% power ultrasonic sonicator, and the shearing was performed according to the ChIP-IT Express shearing Kit (Active motif, 53008). qPCR was used to analyze the binding of HIF-1α to the LDHA promoter.