Ab4830

Cell protein was collected by RIPA lysis buffer (KeyGEN, Nanjing, China) containing 1 nM PMSF (Biotool, Houston, TX, USA). 20 μg protein per well was electrophorsed in 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and incubated with different primary antibodies, including GALNT3 (16716–1-AP, Proteintech, China), caspase3 (ab13847, Abcam, the UK), cleaved caspase3 (ab13585, Abcam, the UK), PARP (ab74290, Abcam, the UK), cleaved PARP (ab4830, Abcam, the UK), MUC1 (ab15481, Abcam, the UK), PI3K-p110α (21890–1-AP, Proteintech, China), p-AKT 308 (AP3743a, Abgent, China), p-AKT 473 (AP3434a, Abgent, China), AKT (ab8805, Abcam, the UK), NF-κB (AP50006, Abgent, China) and GAPDH (AP7873a, Abgent, China), at 4 °C overnight. The membrane was treated with anti-rabbit IgG at 37 °C for 2 h. All bands were detected by an ECL Western blot kit (Thermo Fisher Scientific, USA) and analyzed by Lab Works (TM ver4.6, UVP, Bio Imaging Systems, NY, USA). GAPDH was used as control.

Western blotting was performed in tissue samples or cultured cells as indicated. The cells were lysed in buffer containing 1% NP40, 50 mM Tris, 5 mM EDTA, 1% sodium deoxycholate, 1% SDS, 1% Triton X-100, 10 mg/mL aprotinin, 1 mM PMSF, and 1 mg/mL leupeptin (pH = 7.5), supernatants were collected after spin and protein was measured by Bradford assay (Thermo, Waltham, MA, USA). Forty micrograms total proteins were resolved on SDS-PAGE. Following an electric transfer onto PVDF membranes, the blots with proteins were then blocked by 5% bovine serum albumin and incubated with appropriate primary antibodies at 4 °C overnight. The membranes were incubated by HRP conjugated secondary antibody, and signals were visualized by an enhanced ECL-based imaging system. The caspase 3 detected was active caspase 3. Antibodies used in the study include anti-FOXD3 antibody (ab178512, Abcam, USA), anti-beta Actin antibody (ab8227, Abcam), anti-Annexin A2 antibody (ab41803, Abcam), anti-Cleaved PARP1 antibody (ab4830, Abcam), anti-Casepase-3 antibody (ab13847, Abcam), and anti-GAPDH antibody (ab9484, Abcam). The graphs shown the representative images from three independent experiments. The results were compared with normal human ovarian cell line HOSE.

For Western blotting, protein concentrations in VSMC lysates were quantitated using a BCA Protein Assay Kit (Thermo Fisher Scientific, No. 23225). Proteins (30 μg) were separated through 15% SDS/PAGE and then transferred on to PVDF membranes (Millipore). After 1 h incubation in 5% non-fat milk at room temperature, the blots were further incubated with primary antibodies overnight at 4°C. The primary antibodies used were: anti-Cyclin D1 (Abcam, ab134175), anti-Cyclin E (Abcam, ab33911), anti-cyclin-dependent kinase (CDK) 2 (CDK2) (Abcam, ab32147), anti-CDK4 (Abcam, ab108357), anti-Rb (phospho S807) (Abcam, ab184796), anti-β actin (HC-101-02), anti-active caspase3 (Abcam, ab2302), anti-cleaved poly ADP-ribose polymerase (PARP) (Abcam, ab4830), anti-α-SMA (Abcam, ab32575), anti-SM22α (Abcam, ab14106), anti-Calponin (Abcam, ab46794), anti-Osteopontin (Abcam, ab8448), anti-smoothelin (Abcam, ab21108), and anti-tropomyosin (Abcam, ab181085).
For immunofluorescence, VSMCs were incubated in primary antibodies against α-SMA (Abcam, ab32575) or calponin (Abcam, ab46794).

The transfected TPC-1 and CAL62 were lysed with RIPA buffer (Ding Guo Chang Sheng Biotech, Beijing, China) for 30 min on ice. Following centrifugation at 12,000×g, the concentrations of the cell lysates were measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The cell lysates (30 µg) were analyzed with SDS-PAGE, and electro-transferred onto the PVDF membrane (Thermo Fisher Scientific). The primary antibodies, including anti-LATS1 (ab70561) and anti-p-LATS1 (ab111344) (1:1,500; Abcam); anti-YAP (ab52771) and anti-p-YAP (ab62751) (1:2,000; Abcam); anti-LMO3 (ab230490), anti-LIMK1 (ab39641), and anti-p-LIMK1 (ab194798) (1:2,500; Abcam); anti-Bcl-2 (ab194583) and anti-β-actin (ab8227) (1:3,000; Abcam); anti-cleaved caspase-3 (ab2302) and anti-cleaved PARP (ab4830) (1:3,500; Abcam); anti-cofilin (ab42824) and anti-p-cofilin (ab12866) (1:4,000; Abcam); anti-β-catenin (ab6302), anti-β-tubulin (ab6046), and anti-Histone H3 (ab18521) (1:4,500; Abcam); were used to probe the membranes that were blocked with 5% bovine serum albumin. The membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (ab6721) (1:6,000; Abcam), and the blots were detected by enhanced chemiluminescence (KeyGen, Nanjin, China).

Immunohistochemical analysis was performed as previously described.^{58 (link), 68 (link)} Antibodies were: anti-cleaved PARP (ab4830; 1:50), anti-c-Fos (ab7963; 1:50), anti-c-Jun (ab31419; 1:50), anti-AcH3 (ab1791; 1:200) from Abcam and anti-NTPdase2 (1:100).^{69 (link)} Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Some experiments were performed with anti-c-Fos that had a fluorophore directly attached using the Mix-n-Stain CF Dye antibody labeling kit (Biotium, Hayward, CA, USA). The images were taken via LSCM and we have used single optical section to demonstrate double labeling as well as for cell counting analyses. Identical settings for laser intensity and brightness/contrast were used for comparative analysis between sections.

In this study, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cisplatin were purchased from Alfa and used as received. FITC Annexin V Appoptosis Detection Kit I was purchased from BD Pharmingen™ (Lake Franklin, NJ, USA). Reactive oxygen species assay kit and mitochondrial membrane potential assay kit with JC-1 were obtained from Beyotime. β-Actin (CST, 13E5, #4970), Cyclin B1 (CST, D5C10, #12231), Caspase 3 (CST, D3R6Y, #14220), Cl-PARP1 (Abcam, ab4830), Cl-caspase 3 (Abcam, ab32042), Caspase 9 (Abcam, ab202068), and phospho-CDC2/CDK1 (R&D,Y15) were used as primary antibodies and prepared by 1:1000. HRP AffiniPure Goat Anti-Rabbit (BOSTER) was used as a secondary antibody. CST (Cell Signaling Technology, Danvers, MA, USA).