Caspase 3

The pET28a expression system was obtained from GE Healthcare; the pEGFP-N1 vector was obtained from Clontech; The DNA encoding for segment from 259 amino acids to 345 amino acids of glycoprotein D (AFB76672.1) was amplified by polymerase chain reaction with the primers as following: Sense primer: 5′-GAATTCATGGAGGAGTCGAAGGGC-3′ and anti-sense primer:5′-CTCGAGGATGGCTTCGAGGCTCG-3′, and the DNA fragment was cloning into the pEGFP-N1 vector for construction of the pEGFP-N1-gD, which was used to efficiently express green fluorescent protein (GFP) fused gD protein in 293T cell. Calf antiserum against BoHV-1 was from China Veterinary Culture Collection Center. A mouse anti-His monoclonal antibody (McAb), Alexa Fluor 555- conjugated anti-His antibody, FITC-labeled goat anti-mouse antibody and TRITC-labeled goat anti-mouse antibody were purchased from ThermoFisher Scientific (United States), and a FITC-labeled rabbit anti-bovine antibody was purchased from BioVision (United States). The antibodies against PARP-1, Bcl-2, Bid, caspase-3, caspase-8, caspase-9 and β-actin were purchased from ABclonal Biotech (China). The BoScFv-PE38 was labeled with horseradish peroxidase (HRP) by Sangon Biotech Co., Ltd. (China).

Forty-eight hours after transfection, DF-1 cells were lysed in RIPA buffer (Beyotime, Nantong, China) supplemented with 100 mM phenyl methane sulfonyl fluoride (PMSF) to exact total protein. Protein concentrations were measured by the Pierce BCA Protein Assay Kit (Transgen, Shanghai, China). An equal amount of protein was separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1h. Then, the membranes were separately probed with p-JUN (ABclonal, AP0048), p-FOS (ABclonal, AP0038), p-JNK1 (ABclonal, AP0631), Bcl-2 (ABclonal, A19693), Caspase8 (ABclonal, A0215), Caspase9 (ABclonal, A18676), Caspase3(ABclonal, A19654), GAPDH (Abmart, M20024) overnight at 4°C with a final dilution of 1:5000 (v/v). Finally, the membrane was incubated with the secondary antibody for 1h after TBST washing. The enhanced chemiluminescence (ECL) detection system (Bio-Rad) was used to detect protein expression.

The immunohistochemical staining steps for ki67 and caspase-3 were conducted using their active antibodies ki67 and caspase-3 (ABclonal Technology, Woburn, MA, USA). The staining procedure was at a magnification of 100× in all fields of tissue slices. According to the percentage of Ki67 positive cells (nuclear staining), caspase-3 positive cells (nuclear with or without cytoplasmic staining), and immunohistochemical staining results were scored according to the method described by Sherif et al. [25 (link)]. Regarding Ki67 staining, the basal cell layer staining of seminiferous tubules was excluded (normal proliferation). Immuno-stained slides were image analyzed using Image J software. The staining scores were calculated by the percentage of positive cells within 1000 cells being counted on each slide in the area of maximum staining per 10 high power fields after background subtraction.

RIPA buffer supplemented with 1% PMSF and protease inhibitor cocktail was used to extract protein from both cell lines and tissues. The protein concentrations were measured using BCA assay (Beyotime). Different Samples were separated on 6%–15% SDS‐PAGE gels, followed by protein transfer onto the PVDF membrane (Millipore, USA). After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies against β‐Actin (#8457, CST), GAPDH (#5174, CST), β‐Tubulin (#2146, CST), TAOK3 (#ab70297, Abcam), KMT2C (#28437‐1‐AP, Proteintech), ETV5 (#ab102010, Abcam), IRGM (#AP11128b, Abcepta), H3K4me3 (#9727, CST), LC3B (#ab192890, Abcam), P62 (#ab109012, Abcam), FLAG tag (#66008‐4‐lg, Proteintech), Cleaved PARP (#5625, CST), Caspase‐3 (#A2156, Abclonal), BCL‐2 (#A19693, Abclonal), BAX (#41162, CST), BAK (#A10754, Abclonal), Lamin B (#A11459, Abclonal), P‐KMT2C‐S4588 (#E25915, generated from Abclonal) and P‐KMT2C‐T4589 (#E25804, generated from Abclonal) overnight at 4 °C. After the membranes were washed three times with TBST for 10min each, the membranes were incubated by corresponding HRP‐labeled secondary antibody for 2h at room temperature. Signals were examined using an ECL detection system (Tanon).

Caspase-3 (a rabbit polyclonal antibody purchased from AB clonal Technology, Woburn, MA, USA, Cat. No.: A0214), proliferating cell nuclear antigen (PCNA; a mouse monoclonal antibody, purchased from Novus Biologicals, Centennial, CO, USA, Cat. No.: NB500-106SS), Bax (rabbit polyclonal antibody purchased from Biospes, Chongqing, China, Cat. No.: YPA2175), and Bcl2 (Rabbit polyclonal antibody purchased from Biospes, China, Cat. No.: YPA2275) were used for detecting apoptosis. The data related to the antibodies used in immunohistochemical studies are found in Table 2 Deparaffinized paraffin-embedded sections (5 m) were rehydrated in alcohol, boiled in 10 μM citrate buffer (pH 6.0) for 15 min, and then cooled at room temperature for 20 min. All antibodies were used at a dilution of 1:100 for 30 min. Processing of sections was conducted according to the manufacturer’s instructions using the universal kit (EcnoTek HRP Anti-Polyvalent, DAB; ScyTek Laboratories, Inc., 205 South 600 West, Logan, UT, USA). After the reaction was completed, Mayer’s hematoxylin was used to counterstain the area, which was then dehydrated and covered with DPX [53 (link)].

DMSO was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Dasatinib was purchased from Cell Signaling Technology (CST, Danvers, MA, USA) and dissolved in DMSO. ATO was purchased from the First Affiliated Hospital of Harbin Medical University. c‐ABL1, Bcl‐2, Mcl‐1, Noxa and p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); β‐actin antibody was bought from Sigma‐Aldrich; ERK, p‐ERK, STAT5, p‐STAT5, PI3K, p‐PI3K, AKT, p‐AKT, caspase‐9, caspase‐3, PARP, XIAP, Survivin, Bcl‐w, Bik, Bak, Bad, PUMA, Bid, p21, JNK, p‐JNK, p‐ATF‐2, p‐JUN, IRE1, ASK1 and ATF4 antibodies were purchased from CST; TRAF2 antibody was purchased from ABclonal Biotechnology (Wuhan, Hubei, China); PML and ATF6 were purchased from Abcam (Cambridge, MA, USA).