The rat cardiomyocyte cell line H9c2 (Cell Bank of Type Culture Collection of Chinese Academy of Sciences) was cultured in high glucose DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (cat. no. FB15015; Clark Bioscience). The cells were maintained at 37°C in a humidified incubator (HERAcell 150i; Thermo Fisher Scientific, Inc.) containing 5% CO₂. The medium was refreshed every 2 days and cells were subcultured when cell confluence reached 90%. Cell transfection was performed using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Small interfering (si)RNA [negative control (cat. no. siN05815122147; 50 nM) and siRNA-Fbw7 (cat. no. siG180322050951; 50 nM); Guangzhou Ribobio Co., Ltd.] and plasmids [control green fluorescent protein (GFP) and GFP-Fbw7 plasmid (backbone: pEGFP-N1; cat. no. Fbw7-XM_002729089.5; 1 µg/ml); Wuhan Genecreate Biological Engineering Co., Ltd.] were transfected into H9c2 cells for 48 h prior to subsequent experimentation according to the manufacturer's protocol. The medium was refreshed 8 h after the transfection reagent was first added. The sequence of siRNA-Fbw7 was 5′-GATACATCAATCCGAGTCT-3′.

Different concentrations of H₂O₂ were used to treat cells undergoing different experiments. H₂O₂ concentrations ranged between 0 and 500 µmol/l for H₂O₂ gradient tests, whereas a concentration of 500 µmol/l H₂O₂ was used to treat cells undergoing siRNA transfection. All H₂O₂ treatments were performed in a cell incubator (37°C) and occurred 2 h following transfection with siRNA.

H9c2 cells at a confluence of 90% were subcultured in a 96-well plate to perform the CCK-8 analysis. DMEM/CCK-8 (Beyotime Institute of Biotechnology) reagent mixture (10:1; 110 µl/well) was added to replace the primary DMEM after 2 h of H₂O₂ treatment. Subsequently, after incubation for 2 h at 37°C, the optical density value was determined at a wavelength of 450 nm using a microplate spectrophotometer (Tecan Infinite F50; Tecan Group, Ltd.).

After 2 h of treatment with H₂O₂ at different concentrations, 6-cm culture dishes containing H9c2 cells (90% confluence were collected and lysed in lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) on ice for 40 min. Samples were subsequently centrifuged at 12,000 rpm (16,000 × g) at 4°C for 20 min, and the supernatants were collected. The Bradford assay was performed to determine the protein concentration using Coomassie Brilliant Blue and an infinite f50 spectrophotometer (Tecan Life Sciencies).

For the co-IP experiment, four 10-cm culture dishes seeded with H9c2 cells (90% confluence) were included for each experimental group. Exogenous co-IP was performed on H9c2 cells transfected with GFP-Fbw7 (GFP-Fbw7 group) or control plasmid (IgG group), whereas endogenous co-IP was performed on control (without IgG in the protein sample), treatment (500 µmol/l H₂O₂) and IgG groups. All samples were pre-cleared with 30 µl A/G agarose beads (cat. no. 36403ES03; Shanghai Yeasen Biotechnology Co., Ltd.) for 1 h, and lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), Mcl-1 (3 µl; cat. no. D2W9E; Cell Signaling Technology, Inc.) or control IgG antibody (3 µl; cat. no. sc-2025; Santa Cruz Biotechnology, Inc.), and A/G beads were added and mixed overnight at 4°C. The precipitate was washed in lysis buffer and then centrifuged (700 × g at 4°C for 5 min); this procedure was repeated three times. After discarding the supernatant, 25 µl of 2C loading buffer was added, and then samples were heated in boiling water for 7 min to elute proteins.

SDS-PAGE (10%) was performed using 50 µg (western blotting) and 2,000 µg (co-IP) total protein per sample. Bovine serum albumin (5%; Beijing Solarbio Science & Technology Co., Ltd.) was used to block non-specific binding at room temperature for 1 h following transfer to PVDF membranes, prior to incubation with primary antibodies (4°C, overnight). The following antibodies (1:1,000) were used to detect proteins: Anti-Mcl-1 (cat. no. D2W9E; Cell Signaling Technology, Inc.); and anti-Fbw7 (cat. no. 55290-1-AP), anti-Bax (cat. no. 50599-2-Ig), anti-caspase-3 (cat. no. 19677-1-AP), anti-GFP (cat. no. 50430-2-AP), anti-tubulin (cat. no. 11224-1-AP), anti-β-actin (cat. no. 6000B-1-Ig) and anti-GAPDH (cat. no. 60004-1-Ig; all ProteinTech Group, Inc.). Membranes were then incubated at room temperature for 2 h (1:10,000) with horseradish peroxidase-conjugated anti-rabbit (cat. no. A21020) and anti-mouse (cat. no. A2101) antibodies (Abbkine Scientific Co., Ltd.). The blots were visualized using Tanon™ High-sig ECL Western Blotting Substrate (Tanon Science and Technology Co., Ltd.).

Gray value analysis of western blots and ROS quantification was performed using ImageJ (version 1.8.0; National Institutes of Health); all western blotting data were normalized to constitutive loading controls (gray value ratio of proteins/constitutive proteins) to obtain relative gray values.

H9c2 cells (including siRNA-transfected cells) from 6-well plates (90% confluence) treated with different concentrations of H₂O₂ were digested with 0.25% trypsin (EDTA-free), collected and washed with PBS. A total of 500 µl Annexin V Binding Buffer (diluted to 1X with distilled water) with 3 µl each of Annexin V-FITC and propidium iodide (Apoptosis Detection kit; cat. no. AD10; Dojindo Molecular Technologies, Inc.) were added to cells to measure apoptosis. The staining process was performed at room temperature for 15 min. The results were analyzed using a flow cytometer (BD LSRFortessa™; BD Biosciences) with BD FACSDiva software 4.1 (BD Biosciences), and the apoptotic rate was calculated as the sum of the quadrant (Q)2 and Q4 values.

H9c2 cells were subcultured into a 24-well plate and allowed to reach 70% cell confluence. ROS analysis was performed to detect and measure intracellular oxidative stress. Briefly, 2′,7′-dichloro-dihydrofluorescein diacetate (DCFH-DA; ROS detection assay kit; Beyotime Institute of Biotechnology) was diluted with FBS-free DMEM (1:1,000) and 500 µl diluted DCFH-DA was added to each well of a 24-well plate following transfection with siRNA and 2 h of treatment with H₂O₂. After incubation at 37°C for 1 h, each well was refreshed and washed with FBS-free DMEM three times. The images were captured under an inverted fluorescence microscope (Olympus IX71; Olympus Corporation) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Images of western blots, FCM and ROS were processed by Photoshop CC 2017 (Adobe Systems, Inc.). Bar graphs were generated using GraphPad Prism (version 7.0.4; GraphPad Software, Inc.). Statistical analysis was performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA) and data are presented as the mean ± standard deviation. An independent samples t-test was used to analyze significance between two groups, whereas one-way analysis of variance followed by a least significant differences test was applied to compare datasets containing multiple groups. P<0.05 was considered to indicate a statistically significant difference.

In order to study oxidative stress-induced cell injury, a H9c2 cell injury model was induced following treatment with increasing concentrations of H₂O₂. CCK-8 analysis indicated that the viability of H9c2 cells was reduced as H₂O₂ concentration increased (P<0.05; Fig. 1A). Furthermore, Fbw7 and Bax expression levels, and the cleaved/total caspase-3 ratio were increased, whereas Mcl-1 expression was decreased following treatment with increasing concentrations of H₂O₂, as determined by western blotting (Fig. 1B and C). Flow cytometry revealed that the cell apoptotic rate was markedly increased alongside the intensity of H₂O₂ treatment, and the highest apoptosis level was observed in the 500 µmol/l group (P<0.01; Fig. 1D and E)

To confirm the function of Fbw7 in H9c2 cell injury, Fbw7 expression was knocked down and alterations in oxidative stress-induced injury were observed. Cell viability in the negative control (NC) and siRNA (Si) groups was decreased in response to increasing concentrations of H₂O₂, whereas the viability of the Si group was increased compared with the NC group under the same level of oxidative stress (P<0.05; Fig. 2A). Compared with in the NC + 500 µmol/l H₂O₂ (NC+) group, Fbw7 and Bax expression, and the cleaved/total caspase-3 ratio, were decreased, and Mcl-1 expression was increased in the Si + 500 µmol/l H₂O₂ (Si+) group (Fig. 2B and C). It may be hypothesized that knockdown of Fbw7 facilitated Mcl-1 accumulation and alleviated oxidative stress-induced cell injury. Furthermore, FCM revealed that the apoptotic rate in the Si+ group (37%) was markedly lower compared with in the NC+ group (61.5%; Fig. 2D and E).

ROS analysis was performed to further investigate the mechanism underlying oxidative stress-induced cell injury. The proportion of H9c2 cells exhibiting ROS fluorescence (green staining) was increased as H₂O₂ concentration increased, and the highest fluorescence intensity was detected in the 500 µmol/l group (Fig. 3A). There was no statistically significant difference in ROS fluorescence intensity between the NC and Si groups; however, following treatment with 500 µmol/l H₂O₂, the number of cells with ROS fluorescence was decreased in the Si+ group compared with in the NC+ group (Fig. 3B). These results suggested that oxidative stress-induced cell injury may be mediated by ROS accumulation, and Fbw7 silencing could decrease ROS levels.

Co-IP was used to confirm the interaction between Fbw7 and Mcl-1. For exogenous co-IP, Fbw7 was successfully overexpressed in H9c2 cells transfected with GFP-Fbw7 (data not shown). The results indicated that the exogenous GFP-Fbw7 band was at 130 kDa in the overexpression group, and the binding band of GFP-Fbw7 was shown at the same site. Mcl-1 expression was decreased in response to Fbw7 overexpression (Fig. 4A). For endogenous binding of Fbw7 and Mcl-1, Fbw7-Mcl-1 binding had the tendency of being enhanced under H₂O₂ treatment (500 µmol/l) compared with in the control group (Fig. 4B). These results confirmed the interaction between Fbw7 and Mcl-1, and suggested that decreased Mcl-1 expression may be associated with the E3 ligase function of Fbw7.