Dnmt3b

Total protein was isolated from cells using Pierce RIPA buffer (Thermo Fisher Scientific, 89900) with PMSF protease inhibitor (Sigma-Aldrich, 36978) and run in precast gels (Bio-Rad, 456-1094S). The membrane was blocked with 3% BSA and then probed with primary antibodies against the proteins p-IRF3 (Thermo Fisher Scientific, PA536775), IRF3 (Abcam, ab68481), p-STING (Cell Signaling Technology, 19781S), p-STAT1 (Thermo Fisher Scientific, 33-3400), DNMT3B (Cell Signaling Technology, 67259S), and β-actin (Sigma-Aldrich, A5441), followed by secondary antibodies (anti-mouse or -rabbit IgG, AP-linked, Cell Signaling Technology), washed 3 times, and substrate added (Thermo Fisher Scientific, 45-000-947). They were then developed with a chemiluminescence kit and imaged on an iBright imager (Thermo Fisher Scientific).

The analysis of western blot was described previously [28 (link)]. Briefly, total protein was extracted and applied to SDS-polyacrylamide gels for electrophoresis (22 mA per gel). Protein was transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes for 1.5 h with 400 mA. The transferred membranes were incubated with blocking buffers for 10 min and then incubated with specific primary antibody against E-cadherin (BD), N-cadherin (Genetex), Vimentin (BD), DNMT 1 (Abcam), DNMT 3A (Abcam), DNMT 3B (Cell signaling), or β-Actin (Cell signaling) at appropriate dilutions at 4 °C overnight. The membranes were washed and incubated with horseradish peroxidase-linked secondary antibody for 1 h at room temperature. Bands were visualization by chemiluminescent reagent and record by photographic film and the intensity of the band was quantified and calculated. The results were conducted independently in triplicate.

Evodiamine (purity > 98%) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-aza-2′-deoxycytidine (5-aza) and trichostatin (TSA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Propidium iodide (PI) staining, CD44 and CD133 were purchased from Biosciences (BD Biosciences, NJ, United States). β-actin, E-cadherin, N-cadherin and Vimentin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). cdc2, cyclinB1, DNMT1, DNMT3A, DNMT3B, Histone H3 primary antibodies and HRP-conjugated secondary antibody were obtained from Cell Signaling Technology Inc. (Beverly, MA, United States). Notch3 primary antibody was purchased from Abcam (Cambridge, United Kingdom).

Cultured cells were collected, rinsed with chilled PBS, and lysed in buffer containing 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 150 mM NaCl, 1% sodium deoxycholate, 20 mM Tris–HCl (pH 7.5), 1 mM EGTA, 1% NP-40, 1 mM Na₃VO₄, 1 mM Na₂EDTA, and 1 µg/ml leupeptin. The lysis buffer was supplemented with 1 mM PMSF immediately before cell lysis. Lysates were sonicated and then centrifuged at 13,000 rpm for 15 min at 4 °C to pellet insoluble proteins. Cell lysates were subjected to 8% or 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). Nonspecific binding sites were blocked using 5% non-fat dry milk (Bio-Rad, Hercules, CA) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies specific for DNMT1, DNMT3a, and DNMT3b (Cell Signaling Technology 5425, Danvers, MA) at 4 °C overnight. These membranes were then washed with TBS-T three times (5 min each) and incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using a highly sensitive SuperSignal West Pico Chemiluminescent Substrate (Invitrogen) for detecting HRP.

Total cellular proteins and mouse testicular tissues were extracted using the Total Protein Extraction Kit (Keygentec, China) and protein concentrations were measured using the BCA Protein Assay kit (Pierce, USA). Proteins were run on SDS-PAGE gels, blotted on nitrocellulose membrane, and immunodetected with primary antibodies against PTEN, PI3K, p-PI3K, AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR (S2448), cleaved caspase-3, Bax, Bcl-2, DNMT1, DNMT3a, DNMT3b (Cell Signaling Technology), and γ.H2AX (Abcam). β-actin (Abcam) were detected as controls. Signals were visualized by ECL blotting detection reagents (Thermo Fisher Scientific, USA) and exposed to X-ray films which were scanned and quantitatively analyzed using Image Lab Software (Bio-Rad, USA).