ES2 cell lines and NIH:OVACAR-3 cell lines were purchased from ATCC (Manassas, VA, USA) and RIKEN BioResource Center (Tsukuba, Japan). These ovarian cancer cell lines were routinely cultured in RPMI 1640 (Nacalai Tesque, Kyoto, Japan) containing inactivated 2% fetal bovine serum (Equitech-Bio, Kerrville, TX, USA) and 1% penicillin–streptomycin (Nacalai Tesque) at 37°C with 5% CO2 in room air.
Cell proliferation assay and lactate dehydrogenase (LDH) cytotoxicity assay
We seeded the cells in 12-well plates (100,000 cells/well) in the culture medium. At 80% confluence, the cells were treated with 0–20 µM of bexarotene (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) for 24 hours. The cells were then treated with trypsin–EDTA solution (Nacalai Tesque) and mixed with the total amount of trypan blue solution (Nacalai Tesque). The live cells were counted using a cell-counting chamber (WakenBtech Co., Ltd., Kyoto, Japan) under a stereomicroscope (Olympus, Tokyo, Japan).
Moreover, we collected the cell culture supernatant and measured the LDH activity using the LDH Cytotoxicity Assay Kit (Nacalai Tesque) according to the manufacturer’s protocol. The LDH levels of the cell culture supernatant derived from the bexarotene-treated cell group were normalized relative to the cultured medium derived from the vehicle group. We repeated each experiment at least three times and compared the LDH activity with that of the vehicle group.
Protein extraction and western blot
We washed the cultured cells with PBS and extracted the total protein using a complete Lysis-M reagent (Roche, Basel, Switzerland) containing 1% Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher Science, Waltham, MA, USA) for 15 minutes at room temperature. We subsequently centrifuged the cell lysate at 15,000×g for 15 minutes at 4 °C, separated 7.5 µg of total protein in 10% or 4%–15% gradient SDS-PAGE (Bio-Rad, Hercules, CA, USA), and transferred it to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). Nonspecific binding to the PVDF membrane was blocked in Blocking one-P solution (Nacalai Tesque) at room temperature for 30 minutes. The first antibody was reacted at 4 °C for overnight. Then, we washed the PVDF membrane in PBS-T and enhanced the protein signal by anti-mouse or rabbit IgG antibody (1/10000 or 5000) at room temperature for 1 hour. We used ECL select or ECL Prime reagent (Roche) to detect protein signals.
RNA extraction and Real-time quantitative PCR
Total RNA from cultured cells was extracted using the RNeasy Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Science, Waltham, MA, USA), we reverse-transcribed the extracted RNA into complementary DNA (cDNA). Real-time quantitative PCR (RT-qPCR) was conducted using Light Cycler DNA Master SYBR Green I Kit (F. Hofmann–Roche Ltd., Basel, Switzerland) with Light Cycler Nano System (F. Hofmann–Roche Ltd). Furthermore, we evaluated the CDKN1A and GAPDH (internal control) mRNA expression levels. The expression levels of measured genes were normalized relative to that of GAPDH mRNA. The 2−∆∆Cq method was used to obtain the relative quantitative value (Schmittgen and Livak 2008). Supplementary Table S1 lists the gene-specific primers.
Small interfering RNA (siRNA) transfection
Using Lipofectamine RNAiMAX (Thermo Fisher Science, Waltham, MA, USA), we transfected ES2 cell lines with a siRNA. The si-protein kinase R-like endoplasmic reticulum kinase (PERK; SI02223718; Qiagen, Hilden, Germany) RNA, si-inositol-requiring enzyme 1-A (IRE1A; SI00605255; Qiagen), or a non-target siRNA (control) were used. To prepare the siRNA transfection solution for each tube, we mixed 5 pmol of siRNA with 50 μL of Opti-MEM reduced-serum medium (Thermo Fisher Science, Waltham, MA, USA). Concurrently, we mixed 1 μL of Lipofectamine RNAiMAX with 50 μL of Opti-MEM. Then, the two solutions were mixed by gentle pipetting and incubated for 5 minutes at room temperature to allow siRNA/lipid complexes to form. The treated cells forming siRNA/lipid complexes were incubated for 48 h at 37°C, followed by bexarotene treatment.
Antibodies and other reagents
To detect the target protein, we used anti-caspase-4 antibody (1/1000; Cell Signaling Technology, Danvers, MA, USA), anti-GSDME antibody (1/1000; Proteintech Group, Rosemont, IL, USA), anti-cyclin D1 antibody (1/1000; Cell Signaling Technology), anti-Rb antibody (1/1000; Cell Signaling Technology), anti-phospho-Rb antibody, anti-CDK4 antibody (1/1000; Cell Signaling Technology), anti-CDK6 antibody (1/1000; Cell Signaling Technology), anti-PERK antibody (1/1000; Cell Signaling Technology), anti-Binding immunoglobulin protein (BiP) antibody (1/1000; Cell Signaling Technology), and anti-β actin antibody (1/5000; Cell Signaling Technology) as the first antibodies.
We used ZYVAD-FMK (R&D SYSTEMS, Minneapolis, MN, USA) for caspase-4 inhibition experiments.
The cell proliferation assay was statistically analyzed using an independent t-test. All comparisons were performed using a two-sided test. In addition, P < 0.05 was considered statistically significant. All statistical data were analyzed using the JMP statistical software (SAS Institute Inc., Cary, NC, USA).