The methods used in this study are based on previously published reports (3,17,20,28,29). PHT and 18α-GA were purchased from Sigma-Aldrich, Japan K.K. (Tokyo, Japan). Four primary cultures of fibroblasts derived from the gingiva of healthy donors were obtained from ScienCell™ Research Laboratories (cat. no. 2620, https://sciencellonline.com/human-gingival-fibroblasts/, San Diego, CA, USA). Those cells had been cryopreserved at passage one and delivered frozen. Cells were cultured in an atmosphere of 5% CO₂/95% air maintained at 37˚C in Dulbecco's modified Eagle medium (High Glucose) with L-Glutamine and Phenol Red (D-MEM, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% foetal bovine serum, 50 units/ml penicillin and 50 µg/ml streptomycin (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) until they reached semi-confluence. Cells were routinely passaged using 0.05 w/v% Trypsin-0.53 mmol/l EDTA·4Na Solution with Phenol Red (FUJIFILM Wako Pure Chemical Corporation). Cells were used between passages 6 and 9 for subsequent experiments (Fig. 1). The concentrations of PHT and 18α-GA used in this study were decided according to the results of previous studies as follows: 0.25 µM PHT significantly inhibited the G₁ cell cycle arrest and increased the cell proliferation of gingival fibroblasts compared with the untreated control (17,29); 10 µM 18α-GA significantly decreased the proliferation of gingival fibroblasts compared with 0, 0.1, and 1 µM 18α-GA (20).

Apoptosis assays were performed using an APOPercentage™ Apoptosis Assay Kit (BiocolourLtd., Northern Ireland, UK). After semiconfluent cells were treated with 0.25 µM PHT with or without 10 µM 18α-GA in serum-free D-MEM for 24, 48 and 72 h, the apoptotic cells were labelled with APOPercentage Dye in fresh D-MEM at 37˚C in 5% CO₂ for 1 h. The D-MEM containing the dye was removed, after which the APOPercentage Dye release reagent was added into the cell culture plates and the plates were gently shaken for 10 min. The absorbance of the released dye at 550 nm was then determined. The methods used in this study are based on previously published reports (3,17,20).

The propidium iodide staining and flow cytometric analysis were performed using a CycleTEST™ plus DNA Reagent Kit (Becton Dickinson and Company, Franklin Lakes, NJ, USA; BD). After semiconfluent cells were treated with 0.25 µM PHT with or without 10 µM 18α-GA in serum-free D-MEM for 48 h, cells were harvested by trypsinization, washed three times with Buffer Solution, and then treated with Solution A (trypsin buffer), Solution B (trypsin inhibitor and RNase buffer) and Solution C (PI stain solution) in accordance with the manufacturer's instructions. A BD FACSCalibur™ Flow Cytometer (BD Biosciences) acquired 20,000 events for each sample, and the percentage of cells in the Sub-G₁ (apoptotic), G₀/G₁, S and G₂/M phases of the cell cycle were determined using BD CellQuest Pro Software (version 3.1, BD Biosciences). The methods used in this study are based on previously published reports (3,17,20).

After semiconfluent cells were treated with 0.25 µM PHT with or without 10 µM 18α-GA in serum-free D-MEM for 12 h, total RNA was immediately extracted from the cells using a RNeasy Mini Kit (QIAGEN, Tokyo, Japan). A standard spectrophotometric method was used to assess the concentration and purity of each extracted total RNA. One µg of each total RNA was then reverse-transcribed using a PrimeScript™ RT reagent Kit (TAKARA BIO INC., Shiga, Japan). The cDNAs were analyzed by qPCR in an Eco™ Real-Time PCR System (Illumina, Inc., San Diego, CA, USA) using a KAPA SYBR^® FAST qPCR Master Mix Kit (KAPA BIOSYSTEMS Inc., Wilmington, MA, USA). The following thermocycling conditions were used for qPCR: Enzyme activation at 95˚C for 30 sec, followed by 45 cycles of denaturation at 95˚C for 5 sec and annealing and extension at 60˚C for 20 sec. A Perfect Real Time Support System (TAKARA BIO INC.) was used to synthesize the following PCR primers: B-cell CLL/lymphoma 2 (BCL2); baculoviral IAP repeat containing 3 (BIRC3); CASP8 and FADD-like apoptosis regulator (CFLAR); CASP2 and RIPK1 domain containing adaptor with death domain (CRADD); Fas (TNFRSF6)-associated via death domain (FADD); receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1); tumor necrosis factor receptor superfamily; member 1A (TNFRSF1A); TNF receptor-associated factor 2 (TRAF2); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These primer sequences were used according to the method of Takeuchi et al (3). The PCR primers for Caspases-2, -3, -8, -9 and -10 were synthesized by Custom DNA Oligos (Merck KGaA, Darmstadt, Germany) and Primer-BLAST (National Library of Medicine, Bethesda, MD, USA). Primer sequences used are listed in Table I. Relative quantification was calculated using the 2^-∆∆Cq method (30). After normalization to GAPDH, RNA ratios in treated vs. control cultures were determined. The methods used in this study are based on previously published reports (3).

After semiconfluent cells were treated with 0.25 µM PHT with or without 10 µM 18α-GA in serum-free D-MEM for 24 h, the cells were washed with 37˚C PBS and lysed using β-ME Sample Treatment for Tris SDS (COSMO BIO Co., LTD, Tokyo, Japan). Protein concentrations were determined using a TaKaRa Bradford Protein Assay Kit (TAKARA BIO INC.). Equal quantities of protein extracts (10 µg/lane) were separated via SDS-PAGE with Running Buffer Solution for SDS-PAGE (NACALAI TESQUE, INC., Kyoto, Japan) after which the proteins were transferred to PVDF membranes. The membranes were blocked for 30 min at room temperature with Bullet Blocking One for Western Blotting (NACALAI TESQUE), after which they were probed at room temperature for 1 h with primary antibodies against Caspase-2 (1:500), Caspase-3 (1:1,000), Caspase-9 (1:1,000) and β-Actin (1:1,000). The membranes were washed three times with Tris Buffered Saline with 0.05%-Detergent (NACALAI TESQUE) for 5 min at room temperature, and were then incubated with the secondary antibody (1:10,000) at room temperature for 45 min. The primary and secondary antibodies were diluted using Can Get Signal^® Immunoreaction Enhancer Solution (TOYOBO CO., LTD., Osaka, Japan). After washing, the blots were detected using Chemi-Lumi One Super (NACALAI TESQUE) and ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The densities of western blot bands were measured using ImageJ (1.53t; Java 1.8.0_345 [64-bit]). Primary antibodies against Caspase-3 (cat. no. #9662), Caspase-9 (cat. no. #9502) and β-Actin (cat. no. #4967), as well as anti-rabbit HRP-conjugated IgG were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit monoclonal anti-Caspase-2 antibody (cat. no. ab32021) was purchased from Abcam plc. (Cambridge, UK). The secondary antibody (anti-rabbit IgG, HRP-linked antibody; cat. no. #7074) was purchased from Cell Signaling Technology. The methods used in this study are based on previously published reports (3).

After semiconfluent cells were treated with 0.25 µM PHT with or without 10 µM 18α-GA in serum-free D-MEM for 24 h, Caspase-2, -3 and -9 Colorimetric Assay Kits (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) and a spectrophotometer at 405 nm were used according to the manufacturer's protocols to assess caspase activities. Caspase-2, caspase-3 and caspase-9 were labelled using the synthetic peptide substrates VDVAD-p-nitroanilide (pNA), DEVD-pNA and LEHD-pNA respectively. The methods used in this study are based on previously published reports (3,20,28).

All data are reported as mean ± standard error of the mean (SEM). Statistical analysis was carried out using Welch's t-test. P values <0.05 were considered to indicate a statistically significant difference.

Apoptosis was assessed in gingival fibroblasts after treatment with or without 18α-GA. As shown in Fig. 2, gingival fibroblasts treated with 18α-GA showed a time-dependent increase in the relative number of apoptotic cells compared to the untreated control with significant increases at 48 h (1.5-fold) and at 72 h (1.6-fold).

We analyzed the apoptotic cell population (sub-G₁) and the distribution of cell cycle phases (G₀/G₁, S, and G₂/M) in gingival fibroblasts treated with or without 18α-GA. As shown in Fig. 3, treatment with 18α-GA significantly increased the number of apoptotic cells, however it did not change the distribution of cells in the G₀/G₁, S and G₂/M phase.

We analyzed the effects of 18α-GA treatment of gingival fibroblasts on their mRNA expression levels of apoptotic factors (BCL2, BIRC3, CFLAR, CRADD, FADD, RIPK1, TNFRSF1A, TRAF2, CASP2, CASP3, CASP8, CASP9 and CASP10) using qPCR. As shown in Fig. 4, the treatment of gingival fibroblasts with 18α-GA significantly reduced the BCL2 (0.5-fold) mRNA expression level and significantly increased CRADD (1.7-fold), FADD (7.1-fold), RIPK1 (10.3-fold), TNFRSF1A (7.8-fold) and TRAF2 (13.0-fold) mRNA expression levels. Treatment with 18α-GA also increased the BIRC3 (1.7-fold) mRNA expression level and decreased the CFLAR (0.8-fold) mRNA expression level but not significantly. Treatment of gingival fibroblasts with 18α-GA also significantly increased CASP2 (2.2-fold), CASP3 (2.6-fold) and CASP9 (1.6-fold) mRNA expression levels and increased CASP8 (2.9-fold) and CASP10 (2.7-fold) mRNA expression levels but not significantly.

We analyzed the effects of 18α-GA treatment of gingival fibroblasts on the protein expression of caspases- 2, 3 and 9 using western blot analysis. As shown in Fig. 5, treatment of gingival fibroblasts with 18α-GA significantly increased the protein expression levels of caspase-2 (2.3-fold), caspase-3 (2.7-fold) and caspase-9 (2.8-fold) compared to the levels observed in control cells.

We assessed the effects of treating gingival fibroblasts with 18α-GA on the activities of caspases- 2, 3 and 9. As shown in Fig. 6, treatment with 18α-GA significantly up-regulated the activities of caspase-2 (1.5-fold), caspase-3 (1.5-fold) and caspase-9 (1.7-fold) compared to the levels observed in control cells.