Ab64077

Proteins of cochleas were extracted with cell lysis buffer for Western (P0013, Beyotime, China). Protein concentrations were measured with the BCA Protein Assay Kit (P0013, Beyotime, China). Total protein was separated by 8% SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated with rabbit anti-Dot1l antibody (1:200, ab64077, Abcam, UK) or anti-GAPDH antibody (1:1000, AF1186, Beyotime, China) overnight at 4 ℃ and subsequent HRP-labeled Goat Anti-Rabbit IgG (1:1000, A0208, Beyotime, China) for 1 h at 37 ℃. Immunolabeled bands reacting with a chemiluminescent substrate BeyoECL Star (P0018A, Beyotime, China) were detected and quantified by FUSION-FX7 Spectra (Vilber, France).

Proteins from cornea tissue were extracted and quantified using the bicinchoninic acid method. Then, equal concentrations of protein were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and then transferred to a nitrocellulose membrane. Primary antibodies against Dot1l (ab64077), catalase (ab209211), SOD1 (ab51254), SOD2 (ab68155), p38 (ab31828), p-p38 (ab178867), and β-actin (ab8226) were purchased from Abcam (dilution 1 : 1000). β-Actin was used as a loading control to ensure equal loading. Subsequently, membranes were washed twice with PBS and then incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated immunoglobulin G secondary antibody (1 : 2,000; ZDR 5306, ZDR 5307, ZSGB BIO, Beijing, China) at room temperature for 1 h. Specific bands were then visualized using Immobilon Western Chemiluminescence HRP substrate (Merck Millipore, Darmstadt, Germany). Optical densities were detected using Quantity One software (Bio-Rad, Hercules, CA, USA).

Immunohistochemical staining was performed on tissue microarray (TMA) with antibody against Dot1l (ab64077, Abcam, 1:100 dilution) and proper visualization reagent (DakoEnVision Detection System) as previously described [20 (link)]. Olympus CDD camera, Nikon eclipse Ti-s microscope (×200 magnification) and NIS-Elements F3.2 software were used to record the results. The staining intensity and extent was scored by two independent pathologists without the knowledge of the patients’ outcomes. A semiquantitative H-score, ranged from 0 to 300, was used for each sample by evaluating the staining intensities (0: negative, 1: weak, 2: moderate, 3: strong) and distribution areas (0-100%). Three independent shots with strongest staining were selected for each core and the mean score of the three shots was regarded as the final staining intensity for each sample. The H-score cutoff point for determining tumoral Dot1l high/low expression is 95, which was evaluated by X-tile software (Yale University School of Medicine, New Haven, CT, USA) using minimum p value method [21 (link)].

Western blot was performed as previously described [40 (link)]. The antibodies used were listed as below: DOT1L (ab64077, Abcam), CDK2 (2546, CST), Cyclin A2 (4656, CST), PCNA (13110, CST), GAPDH (51332, CST), c-Myc (5605, CST), H3K79me1 (ab2886, Abcam, Cambridge, MA, USA), H3K79me2 (ab3594, Abcam), H3K79me3 (ab2621, Abcam), and H3 (17168-1-AP, Proteintech, Wuhan, China). Gray ratio of each blot was analyzed by using the Image J ver. 1.46 software and protein/GAPDH or protein/H3 ratio was shown.

Immunohistochemistry was performed on 5 μm thick paraffin-embedded EDTA-decalcified knee sections. Heat induced epitope retrieval was performed using a Citrate-EDTA buffer (pH 6.2) for 10 minutes at 95°C. Sections were treated with 3% H₂O₂/methanol for 10 minutes to inactivate endogenous peroxidase, blocked in goat serum for 30 minutes, and incubated overnight at 4°C with primary antibodies against DOT1L (Abcam, ab64077, 6 μg/ml) or HIF1A (Abcam, ab82832, 10 μg/ml) or for 90 minutes with primary antibody against H3K79me2 (Abcam, ab3594, 1 μg/ml). Rabbit IgG (Santa Cruz, sc-2027) was used as negative control. Avidin-biotin complex amplification (Vectastain ABC kit, Vector Laboratories) was used, except for the immunohistochemical detection of H3K79me2. Peroxidase goat anti-rabbit IgG (Jackson Immunoresearch) was applied for 30 minutes, and peroxidase activity was determined using DAB. Images were taken using an Olympus IX83 microscope. Quantification of the DAB staining was performed with a color deconvolution plugin (Jacqui Ross, Auckland University) in ImageJ Software (NIH Image). Quantification was performed using the average of 2 technical replicates for 5 different mice per condition, with staining intensity reported relative to the average of the 5 SHAM+Vehicle mice.