Cyclin a2

For Western blotting analyses, the collected cells were rinsed in PBS and lysed in NETN lysis buffer (Bethyl Laboratories, Inc., Montgomery, TX, USA). Equal amounts of protein were separated on 10%–12% SDS-polyacrylamide gels, transferred to PVDF membranes, and blocked with 5% nonfat dry milk in TBST for 1 h. The membranes were then incubated with primary antibodies overnight at 4 °C. Antibodies against PARP, E-cadherin, N-cadherin, vimentin, cyclin A2, cyclin D1, CDK2, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA) (all used at 1:1000 dilutions). Antibodies against cytochrome c were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) (1:2000 dilutions). After being washed 3 times with TBST for 10 min, the membranes were incubated with appropriate secondary antibodies in TBST for 1 h. After several washes of TBST, the blots were developed by enhanced chemiluminescence (ECL) solution (Bio-Rad, Hercules, CA, USA); Western blots were quantified using ImageJ software (1.8.0_112, Windows).

After denaturation in Laemmli sample buffer, proteins were separated by electrophoresis in SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (BioRad) using Trans-Blot Turbo system (BioRad). Membranes were blocked for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C: cyclin A2 (Cell Signaling, #4656, 1:2,000), cyclin B2 (Santa Cruz, sc-245, 1:1,000), cyclin E1 (Cell Signaling, #4129, 1:1,000), GAPDH (Cell signaling, #2118, 1:1,000) and p21 (Cell Signaling, #2947, 1:1,000). Subsequently, the membranes were washed and incubated with the corresponding anti-mouse/rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse IgG, Dako, 1:2,000 and anti-rabbit IgG, Pierce Biotechnology, 1:25,000). Detection was performed by chemiluminescence using a detection kit from Amersham (ECL) and the Fusion FX imaging system (Vilber Lourmat).

Foetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Thermo Fisher Scientific (Waltham, MA) and 4′,6-diamidino-2-phenylindole (DAPI) was from Sigma-Aldrich (Seelze, Germany). ER-tracker Red was obtained from Thermo Fisher Scientific (Waltham, MA). Mouse monoclonal antibodies (mAbs) against PERK, phosphorylated-eIF2a, CHOP, XBP-1s, cyclin A2 and Cdc25C were obtained from Cell Signaling Technology (Danvers, MA); and anti-β-actin and CDK1 were from Boster (Pleasanton, CA). Salubrinal was purchased from Calbiochem (San Diego, CA).

Cells (VCaP, VCaP‐C, VCaP‐ENZR, C4‐2B, and C4‐2B‐ENZR) were plated in 6‐well plates in ENZ‐free media for 2 days prior to harvesting for protein isolation. For experiments for cell cycle protein, C4‐2B‐ENZR cells were plated in 6‐cm dishes and treated with vehicle, ENZ (20 µm), EPI‐7170 (2.5 or 3.5 µm), or its combination in phenol red‐free RPMI‐1640 plus 1.5% FBS for 48 h and harvested for protein isolation. Protein was extracted with RIPA buffer and protease inhibitor cocktails (Roche Diagnostics, Laval, QC, Canada). The amount of protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of denatured proteins were separated by SDS/PAGE gels and transferred to a PVDF membrane and blocked with 5% skim milk in PBS containing 0.1% Tween 20. The membranes were probed with the following primary antibodies: AR (N‐20) (ab108341) (Abcam, Cambridge, MA, USA), AR‐V7 (RM7) (RevMAb Biosciences, South San Francisco, CA, USA), Cyclin D1 (#2978) diluted 1 : 1000, Cyclin A2 (#4656) diluted 1 : 1000, CDK4 (#12790) (Cell Signaling Technology, Danvers, MA, USA) diluted 1 : 1000, and Actin (A5441) (Sigma‐Aldrich, Oakville, ON, Canada) diluted 1 : 10 000.

The following antibodies were used for IF and diluted in TBS supplemented with 0.1% Tween20 and 2% BSA: CyclinA2 (1:400; #sc-751; Santa Cruz), CyclinA2 (1:400; #4656; Cell Signaling), PLK1 (1:400; ab14210; Abcam), pTCTP (1:400; #5251; Cell Signaling), pH2AX (1:800; #2577; Cell Signaling), pCyclinB1 (1:400; #ab55184; Abcam), pLaminA/C (1:400; #2026; Cell Signaling), Alexa Fluor 488-Goat anti-Rabbit (1:2000; #A11008 Life Technologies) and Alexa Fluor 555-Goat anti-Mouse (1: 2000; #A21422 Life Technologies). The following antibodies were used for WB and diluted in TBS supplemented with 0.1% Tween20 and 5% milk powder: CDC6 (1:400; #sc-9964 Santa Cruz), CDT1 (1:400; #sc-28262 Santa Cruz), GAPDH (1:20000; #G8795; Sigma), anti-Rabbit HRP (1:2000; #ab6721; Abcam) and anti-Mouse HRP (1:2000; #ab97023; Abcam).

Total protein extracts were obtained using the previously described lysis buffer 20 (link). Twenty μg of protein per sample were loaded in 8%, 10% or 15% SDS-PAGE polyacrylamide gels, and then transferred onto PVDF membranes, followed by immunodetection using appropriate antibodies. Antibodies against the following proteins were used: SOX2 (sc-365823; 1:1000), NANOG (sc-293121; 1:1000), MAD2 (sc-28261; 1:1000), BUBR1 (sc-47744; 1:1000), Cyclin B1 (sc-166757; 1:1000) and β-ACTIN (sc-47778; 1:1000) were purchased from Santa Cruz Biotechnology; GLI1 (#3538 1:1000), NOTCH1 (#3608; 1:1000), Cyclin A2 (#4656; 1:1000), Cyclin D1 (#2926; 1:1000), phospho-CDK1^Tyr15 (#9111; 1:2000), CDK1 (#9112; 1:1000) and phospho-Histone H3^Ser10 (#3377; 1:1000) were all purchased from Cell Signaling Technology; Antibody to β-catenin (610154; 1:1000) was acquired from BD Transduction Laboratories and finally, α-Tubulin (1:10,000) from Sigma. Secondary antibodies conjugated with horseradish peroxidase were purchased from BioRad, and chemiluminescence detection was performed using ECL (Santa Cruz Biotechnology).