Kdm5a

Total protein was extracted from cancer cells and tissues using radio-immunoprecipitation assay lysis buffer, with the protein concentration then determined with a bicinchoninic acid kit. The protein was separated and transferred onto a polyvinylidene fluoride membrane. The membrane was then blocked and underwent overnight incubation with primary antibodies against KDM5A (1:10000), H3K4me3 (1 μg/mL, rabbit, Abcam, USA), YTHDF2 (1:2000, rabbit) and MOB3B (1:1000, rabbit, Sigma-Aldrich Chemical Company, St Louis, MO, USA). The next day, the membrane was re-probed with horseradish peroxidase-labeled secondary goat anti-rabbit IgG (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Afterwards, the membrane was visualized and the protein bands were quantified using the Bio-Rad ChemiDoc™ system and ImageJ2x software. The ratio of the gray value of the target band to GAPDH (1:10000, rabbit, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was representative of the relative protein expression.

Formalin‐fixed paraffin‐embedded 5‐μm tissue sections were incubated at 60°C for 30 minutes and then deparaffinized in xylenes, rehydrated through a graded series of alcohol concentrations for 5 minutes and rinsed by water for 2 minutes. After antigen retrieval by 1mM Tris‐EDTA (pH = 8.0) was performed, all sections were rinsed with phosphate buffer saline (PBS) for three times and left to block at room temperature in 3% H₂O₂‐methanol for 10 minutes, followed by the coating of antibodies at room temperature for overnight at 4℃. Afterwards, the sections were rinsed three times in 0.1% PBST, incubated with an Enhancer buffer (PV‐9000, Zsbio, Beijing, China) for 20 minutes and incubated with enzyme‐labelled anti‐mouse/rabbit secondary antibodies (PV‐9000, Zsbio) for 30 minutes. Subsequently, the proteins were subjected to diaminobenzidine (DAB) development and left to counterstain with haematoxylin for 1 minute. After the differentiation and addition of blue coloration, the sections were dehydrated by gradient concentration ethanol, transparentized and sealed by neutral resins. Immunohistochemistry images were obtained using an upright microscope (BX53, OLYMPUS, Japan) and analysed and scored by experienced pathologists. Primary antibodies used: KDM5A (1:400, Abcam, Cambridge, UK, ab217292) and CD31 (1:400, Abcam, ab9498).

The total cellular proteins were extracted with RIPA lysis buffer, and 20 mg were used. The membranes were probed with antibodies against p16^INK4 and p27^kip1 from Santa Cruz Biotechnologies, H3K4me3 (1:1000), H3K4me2 (1:1000), GAPDH (1:1000), and KDM5A (1:2500) from Abcam (Cambridge, MA, USA), followed by anti-mouse or rabbit horseradish peroxidase-conjugated immunoglobulin (Ig) G (1:1000) and developed with the enhanced chemiluminescent method. GAPDH signal served as a loading control. For immunoblotting of histone H3K4 mono-, di- and tri-methylation, we isolated a total histone fraction from nuclei using dilute acid extraction. Two micrograms of histone proteins were used and detected with antibodies against di- and tri-methylated H3K4 (Millipore, Billerica, MA, USA).

Protein expression levels in whole cell extracts were determined as previously described [41 (link)]. Primary antibodies used were: Fli1 (Abcam, ab15289, 1:1000); KDM5A (Abcam, ab70892, 1:500); PHF2 (Cell Signaling Technology, #3497, 1:1000); L1CAM (Cell Signaling Technology, #89861S, 1:1000) and α-tubulin (Sigma, #T5168, 1:20,000). For histone mark analysis, harvested cells were resuspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.02% (w/v) NaN₃) at a density of 10⁷ cells/ml and lysed for 10 minutes on ice. The resulting suspension was spun 10 minutes at max speed at 4°C to pellet nuclei. Nuclei were washed in one half of the original volume of TEB, collected by centrifugation as before, resuspended in 0.2 N HCl at a density of 4 × 10⁷ nuclei/ml, and rotated overnight at 4°C to extract histones. Extracted histones were collected by spinning 10 minutes at max speed at 4°C, and collecting the supernatant. Histone extracts were subjected to SDS-PAGE and immunoblotting as previously described [20 (link)]. Primary antibodies used were: H3K4me3 (Cell Signaling Technologies, #9751, 1:1000) and H3 (Abcam, #1791,1:1000).