Celastrol, sodium tauroursodeoxycholate (TUDCA), thapsigargin (TG) and Hoechst 33258 were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Celastrol had a purity higher than 98%. A stock solution (50 mmol/l) was made in dimethyl sulfoxide (DMSO) (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) and stored in the dark at −80°C. GSK2656157 was purchased from Selleck Chemicals (Houston, TX, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and trypsin were all purchased from Gibco™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell viability and cytotoxicity test kits [Cell Counting Kit-8 (CCK-8)] and FLUOU-3/AM were obtained from Dojindo Molecular Technologies (Kumamoto, Japan). The Annexin V-propidium iodide (PI) double-staining test kit was purchased from Nanjing KeyGen Biotech. Bip, Calnexin, endoplasmic oxidoreductin-1-like protein α (Ero1-Lα), protein disulfide isomerase (PDI), IRE1α, PERK, CHOP, cleaved caspase-3, cytochrome c, Bax, Bcl-2, LC-3, p62 and β-actin were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Cleaved caspase-12 was purchased from Proteintech Group Inc., (Wuhan, China) and p-PERK was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). HOS cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in DMEM containing 100 µg/ml penicillin, 100 µg/ml streptomycin, and 10% FBS (complete medium) at 37°C in 5% CO₂.

The anti-proliferative effect of celastrol on HOS cells was assessed by after 12 h, they were treated with various concentrations of celastrol (0, 1.5, 2.5, 3.5 and 4.5 µM) for 24 h. Subsequently, 10 µl CCK-8 was added to each well, and the plates were incubated in an incubator for 1 to 2 h at 37°C following the manufacturer's instructions. A microplate reader was used to detect the absorbance at 450 nm. Data represented the mean of five replicates. The cell viability was calculated using the following equation: Cell viability (%) = Average OD in study group/average OD in control group ×100%, where OD was the optical density. Each assay was performed in triplicate. Based on the results of the cell viability assay, we selected an appropriate celastrol concentration for subsequent experiments.

HOS cells were seeded in 24-well plates at a density of 5×10⁴ cells/well, and treated with the celastrol concentration of 3.00 µM for 24 h. Subsequently, the culture medium was discarded, and the cells were washed three times with phosphate-buffered saline (PBS) (Nanjing KeyGen Biotech), then fixed with 1% osmic acid and 2.5% glutaraldehyde, dehydrated using acetone and gradient ethanol, embedded, solidified, sliced using an ultramicrotome and stained with 3% uranium acetate-lead citrate. The cells were observed to evaluate morphological changes and images were captured with transmission electron microscopy.

HOS cells were seeded in 24-well plates at a density of 5×10⁴ cells/well, and treated with the celastrol concentration of 3.00 µM for 24 h. Subsequenlty, the culture medium was discarded, and the cells were washed three times with PBS. After the HOS cells were fixed with 4% paraformaldehyde for 5 min, 10 µg/ml Hoechst 33258 (200 µl) was added to the cells at 37°C for 5 min in the dark. The cells were observed to evaluate apoptotic changes and images were captured with a fluorescence microscope after being washed three times with PBS.

HOS cells were seeded in 6-well plates at a density of 5×10⁶ cells/well. Suspended and adherent cells were collected after treatment and analyzed with an Annexin V/PI apoptosis kit using FCM.

HOS cells were seeded in 6-well plates at a density of 5×10⁶ cells/well. Suspended and adherent cells were collected following treatment and analyzed with a FLUO-3/AM (2 µM) kit using FCM.

HOS cells were seeded in 24-well plates at a density of 5×10⁴ cells/well, and treated with the celastrol concentration of 3.00 µM for 24 h. Subsequently, the cells were washed with PBS, fixed in 4% freshly prepared formaldehyde for 5 min, and permeabilized in PBS with 0.1% Triton X-100 for 5 min at room temperature. Cells were then incubated for 1 h in blocking solution (3% bovine serum albumin/0.08% glycine in PBS) and treated with a rabbit polyclonal antibody against p-PERK (1:1,000; cat. no. Sc-32577; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at room temperature. The secondary antibody, fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit (1:200; cat. no. SA00003-2; ProteinTech Group, Inc., Chicago, IL, USA), was applied for 1 h at room temperature, and nuclei were counterstained with DAPI (Beyotime Institute of Biotechnology, Shanghai, China). Slides were viewed under a fluorescence microscope (Axio Observer A1; Carl Zeiss AG, Oberkochen, Germany).

Western blotting was performed to observe the protein expression level. Briefly, at various time-points, cells in the control and celastrol groups were collected and lysed using radioimmunoprecipitation assay (RIPA) buffer (Keygen Biotech, Jiangsu, China) containing protease inhibitor cocktail (Selleck Chemicals, Houston, TX, USA) for 30 min on ice. The following steps were performed as depicted in our previous study (11).

The quantitative data are represented as the mean ± standard deviation (SD) and comparisons of significant differences were calculated by one-way analysis of variance (ANOVA) with Dunnett's test or unpaired Student's t-test. All statistical analyses were performed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). Statistical significance tests were two-tailed, and P<0.05 was considered to indicate a statistically significant difference.

The effect of celastrol on cell viability was investigated by exposing HOS cells to various concentrations of celastrol for 24 h (Fig. 1). The CCK-8 assay demonstrated that celastrol could inhibit the viability of HOS cells in a dose-dependent manner and revealed that the 50% inhibitory concentration (IC₅₀) was 3.36 µM for HOS cells. Therefore, based on previous experiments, we chose a celastrol concentration of 3.00 µM as the treatment condition for the following experiments (10).

Under inverted phase contrast microscopy, retracted, round and floating cells were observed, and karyopyknosis, condensation, karyorrhexis and other typical apoptotic bodies were observed with Hoechst 33255 nuclear staining following treatment with celastrol for 24 h (Fig. 2A and B). However, there were no significant changes in the control group. FCM revealed that the apoptosis rates of the 6, 12 and 24-h groups were all higher than that of the control group (P<0.05) (Fig. 2C and D). These results indicated that celastrol induced apoptosis of HOS cells.

Under Transmission electron microscopy (TEM), the HOS cells displayed swelling of the ER following celastrol treatment for 24 h, whereas the control group exhibited no changes in the ER (Fig. 3A). FCM demonstrated that the level of calcium in the celastrol (24 h) group was significantly higher than that in the control group (Fig. 3B). In addition, western blotting revealed that at 6, 12 and 24 h following treatment using celastrol, the expression levels of ERS-related proteins (Bip, p-PERK, IRE1α, Calnexin, Ero1-Lα and PDI), ERS-related apoptosis proteins (CHOP, cleaved caspase-12) and cleaved caspase-3 in HOS cells were all increased, while PERK was decreased compared to those of the control group (Fig. 3C and D, Fig. 4A and B; P<0.05). These results indicated that ERS was induced by celastrol in HOS cells, and ERS-induced apoptosis was also involved in HOS cell apoptosis induced by celastrol. To exclude false-positive results of the experiment and the influence of DMSO, following treatment with TG, we observed that the expression levels of Bip, CHOP and cleaved caspase-12 in HOS cells all increased, while following treatment with DMSO, all of them decreased, indicating that TG could induce ERS-induced apoptosis but DMSO could not (Fig. 4C and D; P<0.05).

Furthermore, western blotting also revealed that following treatment with celastrol for 6, 12 and 24 h, Bax and cytochrome c in the cytoplasm, indexes of mitochondrial apoptosis, were increased, while Bcl-2 was decreased (Fig. 5A and B; P<0.05). These findings indicated that the mitochondrial pathway was involved in celastrol-induced apoptosis in HOS cells.

TUDCA (500 µM), an inhibitor of ERS, was used to pretreat HOS cells for 1 h before celastrol treatment (17,18). FCM demonstrated that pretreatment with TUDCA significantly increased the apoptosis rate compared to celastrol treatment alone (Fig. 6A and B; P<0.05). Furthermore, western blotting revealed that pretreatment with TUDCA promoted the expression of Lc-3 II and cleaved caspase-3 compared with celastrol alone treatment, while the expression of Bip, p-PERK, and p62 was decreased (Fig. 6C and D; P<0.05). All the results indicated that attenuating the ERS of HOS cells would promote the autophagy and apoptosis induced by celastrol.

The unfolded protein response (UPR) protects HOS cells from apoptosis induced by celastrol. To maintain homeostasis of HOS cells, a large number of unfolded proteins activate the PERK signaling pathway by binding to Bip, and PERK, which is an important transducer, activates downstream molecular partners and protein secretion related to molecular gene transcription. To explore whether attenuating the expression of PERK signaling pathway members would reverse the protective effect of ERS induced by celastrol and further promote the apoptosis of HOS cells, we used an inhibitor of PERK (GSK2656157, 5 mM) to pretreat HOS cells for 1 h before celastrol treatment (19,20). Following treatment with GSK2656157, the proliferation of HOS cells was further inhibited compared with that of the celastrol group; karyopyknosis, condensation, karyorrhexis and other typical apoptotic bodies were more evident; and the apoptosis rates further increased, while immunofluorescence revealed that the expression of p-PERK was significantly attenuated (Fig. 7A-D; P<0.05). Following pretreatment with GSK2656157, the expression of PERK was inhibited, while cleaved caspase-3 was increased. All these results demonstrated that inhibiting the expression of PERK strongly promoted apoptosis induced by celastrol. In addition, we found that pretreatment with GSK2656157 attenuated autophagy and suppressed the expression of PERK (Fig. 7E-F; P<0.05).