This study was approved by the Institutional Ethics Committee of the Hunan Provincial People's Hospital and in accordance with the Ethical Management Guidelines of Hunan Provincial People's Hospital (approval no. 210139). A total of 32 bladder cancer tissue specimens and matched normal specimens were collected from Hunan Provincial People's Hospital. Informed written consent was obtained from all the participants.

T24 and 5637 cells (American Type Culture Collection) were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 mg/ml streptomycin, and 100 units of penicillin. All the cells were cultured in a humidified incubator at 37°C, supplied with 5% CO₂, 21% O₂, and 74% N₂.

Cells were first seeded in six-well plates and cultured until they reached 30–50% confluence. siRNAs were introduced to the cells through transfection. Cells were transfected with Lipofectamine^® 3000 Transfection Reagent (Beyotime Institute of Biotechnology). For the present study, six groups of cells were used: i) Lipofectamine^® 3000 Transfection Reagent alone; ii) Lipofectamine^® 3000 Transfection Reagent combined with 50 nM/µl NC siRNA; iii) Lipofectamine^® 3000 Transfection Reagent combined with 50 nM/µl si-PSMD14 (5′-AAGGTTGTTATTGATGCCTTCAGAT-3′); iv) Lipofectamine^® 3000 Transfection Reagent combined with 50 nM/µl si-NC for PSMD14 (5′-AAGTTGTGTTACGTACTTCATGGAT-3′); v) Lipofectamine^® 3000 Transfection Reagent combined with 50 nM/µl si-Drp1 (5′-AAGTGGTGACTTGTCTTCTTCGTAA-3′); vi) Lipofectamine^® 3000 Transfection Reagent combined with or 50 nM/µl si-NC for DRP1 (5′-AAGAGTGGTTCTTCTCTTCGGTTAA-3′). Cells were transfected for 24 h before subsequent experiments. Drp1 agonist Mdivi-1 (50 mg/ml; Beyotime Institute of Biotechnology) was also used to manipulate Drp expression. The manufacturer's instructions were followed during the transfection process, and the efficiency of the transfection was confirmed using qPCR.

The bladder tissue sections were treated first by removing the paraffin using xylene and ethanol, and then performing antigen retrieval. Following this, tissues were blocked using 3% BSA and then stained using two different types of antibodies anti-PSMD14 (cat. no. 4197S; Cell Signaling Technology, Inc.; 1:100) or anti-Drp1 (cat. no. 8570S; Cell Signaling Technology, Inc.; 1:100) at 4°C overnight. To ensure the reliability of the results, a control was also included by using mouse serum at the same protein concentration as the antibody solution. Under a light microscope at an ×100 magnification, the positively stained tissue sections could be seen as appearing brown or tawny-brown in color. Images of the tissue were taken using an optical microscope, and the integrated optical density (IOD) values of the tissue sections were then measured using Image-Pro Plus version 6.0 (Media Cybernetics, Inc.). To ensure accuracy, five different fields of view were selected from each tissue section, and three tissue sections were used for each group to determine the positive IOD values. The mean IOD values were then used to calculate the relative expressions of PSMD14 and Drp1.

Cells that had been grown on slides were fixed with 4% paraformaldehyde solution for 15 min at room temperature, and then permeabilized with 0.1% Triton X-100 at room temperature for 15 min. The samples were washed three times with PBS and then sealed with a 5% BSA solution at room temperature for 45 min. Next, the primary antibody was incubated with the sample overnight at 4°C. The primary antibodies used were: Anti-PSMD14 (cat. no. 4197S; 1:200; Cell Signaling Technology, Inc.), anti-Drp1 (cat. no. 8570S; 1:200; Cell Signaling Technology, Inc.), or anti-TOM20 (cat. no. 42406S; 1:200; Cell Signaling Technology, Inc.). DAPI (cat. no. 4083S; Cell Signaling Technology, Inc. 1:200) was used to counterstain the nuclei. The secondary antibodies included Alexa FluoR 555 goat anti-rabbit (cat. no. A27017; 1:400; Invitrogen; Thermo Fisher Scientific, Inc.). A total of five different fields of view were selected and randomly scored using a fluorescence microscope (magnification, ×40, Olympus Corporation).

Cells were collected and lysed using RIPA buffer (Beyotime Institute of Biotechnology) for 30 min at 4°C. The nuclear protein was collected using cytoplasmic and nuclear extraction kit (cat. no. A37487; Invitrogen; Thermo Fisher Scientific, Inc.). The protein lysates were quantified by BCA Protein Assay Kit (Beyotime Institute of Biotechnology). SDS gels (8–12%) were used for SDS-PAGE. The resolved proteins were then transferred to PVDF. To block the membranes, a 10% BSA solution was dissolved in TBS buffer containing 0.05% Tween 20 and added to the membranes at room temperature for 1 h. The primary antibody was then added to TBS containing 0.05% Tween 20 and incubated for at least 10 h at 4°C. The primary antibodies used were anti-PSMD14 (cat. no. 4197S; Cell Signaling Technology, Inc.; 1:1,000) and anti-Drp1 (cat. no. 8570S; Cell Signaling Technology, Inc.; 1:1,000). To ensure accuracy, the protein content was normalized using GAPDH (cat. no. 5174S; Cell Signaling Technology, Inc.; 1:8,000) as the internal control. After washing the membrane three times, the anti-rabbit second antibody (cat. no. 7074S; Cell Signaling Technology, Inc.; 1:8,000) and anti-mouse mice secondary antibody (cat. no. 7076S; Cell Signaling Technology, Inc.; 1:8,000) in TBS containing 0.05% Tween 20 were used at room temperature for 1 h. After three additional washes, the bands were detected by enhanced chemiluminescence and autoradiography. The experiments were repeated three times, and the results were evaluated using Image Lab 6.0 (Bio-Rad Laboratories, Inc.). Drp1 agonist Mdivi-1 (50 mg/ml; Beyotime Institute of Biotechnology) was also used to manipulate the expression of Drp1. The blots were grouped and cropped from the same gel.

Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA purity was assessed using a spectrophotometer. First-strand cDNA was synthesized using a cDNA synthesis kit (Promega Corporation) at 65°C for 5 min according to the manufacturer's protocol. Subsequently, cDNA was amplified by qPCR using an Applied Biosystems SYBR Green mix kit and the ABI 7900 Real-Time PCR system (both Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. qPCR was performed as follows: Denaturation at 94°C for 4 min; followed by 40 cycles of amplification at 94°C for 30 sec, hybridization at 56°C for 30 sec, and extension at 72°C for 30 sec. Quantification of mRNA was automatically conducted using SDS version 1.3 software (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the 2^−∆∆Cq method was employed to calculate mRNA expression (28,29). The sequences of the primers used were: PSMD14 forward, 5′-GTCAGTGTGGAGGCAGTTGATC-3′, and reverse, 5′-CCACACCAGAAAGCCAACAACC-3′; DRP1 forward, 5′-TCACCCGGAGACCTCTCATTC-3′ and reverse, 5′-GGTTCAGGGCTTACTCCCTTAT-3′, and β-actin forward, 5′-CGAGCGCGGCTACAGCTT-3′ and reverse, 5′-TCCTTAATGTCACGCACGATTT-3′.

T24 and 5637 cells in the cell culture flask were digested with 0.25% trypsin. Then these cells were seeded in a 96-well culture plate at a density of 1×10⁴ cells/ml for 24 h. After 24 h of incubation, 10 µl CCK-8 reagent was added to each well for 2 h. The optical density was measured at a wavelength of 450 nm using a Bio-Rad 680 microplate reader. Each experiment was performed in triplicates.

Cells were transfected with si-PSMD14. After transfection for 48 h, the cells were treated with MG132 (15 µm; Beyotime Institute of Biotechnology) overnight and then lysed with RIPA lysis buffer, follow by passing 5 times through a 21-gauge needle. Whole-cell extract (1 mg) was used for immunoprecipitation with 1 µg anti-PSMD14 (cat. no. 4197S; Cell Signaling Technology, Inc.) or anti-Drp1 (cat. no. 8570S; Cell Signaling Technology, Inc.). The proteins that had been pulled down were subjected to SDS/PAGE for immunoblot analysis.

Each experiment was repeated at least three times. The categorical data were assessed using a Fisher's exact test, and the quantitative data are presented as the mean ± SEM. All statistical analyses were performed using SPSS version 19.0 (IBM Corp.). The differences between groups were compared using a Student's t-test or ANOVA followed by a post hoc Dunnett's test or Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

To reveal the potential function of PSMD14 in cancer, immunohistochemical staining and western blotting were used to determine the expression of Drp1 and PSMD14 in bladder cancer tissues, and it was found that compared with normal tissues, Drp1 and PSMD14 expression were abnormally upregulated in bladder cancer tissues (Fig. 1). These results suggest that Drp1 and PSMD14 may play important roles in the occurrence and development of bladder cancer.

The expression of PSMD14 of T24 and 5637 cells transfected with PSMD14 siRNA was detected by western blotting (Fig. 2A). Then cell viability assay and colony formation assays were used to detect the proliferation and colony formation of the transfected bladder cancer cells compared with the respective control cells. The results showed that the knockdown of PSMD14 significantly reduced the growth and proliferation capacity of T24 and 5637 cells (Fig. 2B and C). Mitochondrial fusion in the bladder cancer cells was assessed to explore the relationship between PSMD14 and mitochondrial dynamics. PSMD14 knockdown did not affect the expression of mitochondrial fusion factors (OPA1, Mfn1, and Mfn2) by immunofluorescent staining, and the levels of mitochondrial factor-Drp1 were downregulated by PSMD14 knockdown (Fig. 2D). Drp1 induced mitochondria to produce several fragments, and PSMD14 knockdown blocked this process (Fig. 2E).

Next, whether endogenous Drp1 interacted with endogenous PSMD14 was assessed. Endogenous Drp1 and PSMD14 proteins were co-immunoprecipitated (Fig. 3A), and this finding was confirmed through reciprocal co-IP test (Fig. 3B). Then, si-PSMD14 transfected T24 and 5637 cells were treated with exogenous PSMD14. The results showed that the PSMD14 resulted in an increase in Drp1 in T24 and 5637 cells (Fig. 4A and B).

The results of western blotting results showed that Drp1 was significantly downregulated in T24 and 5637 cells transfected with si-Drp1 (Fig. 5A). Then cell viability and colony formation assays were used to detect the proliferation and colony formation ability of Drp1 knockdown bladder cancer cells. The results showed that the knockdown of Drp1 significantly reduced the growth and proliferation capacity of T24 and 5637 cells (Fig. 5B and C). Additionally, Drp1 knockdown significantly increased mitochondrial length and inhibited mitochondrial fission (Fig. 5D).

The above results confirmed that Drp1 plays an important role in PSMD14-mediated bladder cancer. Next, whether the upregulation of Drp1 reversed the anticancer effects mediated by PSMD14 knockdown was assessed. Drp1 agonist Mdivi-1 (50 mg/ml) was used to upregulate Drp1 in bladder cancer cells (Fig. 6A). Then the proliferation of si-PSMD14 transfected bladder cancer cells treated with Mdivi-1 was determined. The results of the cell viability assays showed that Mdivi-1 significantly increased the growth of T24 and 5637 cells transfected with si-PSMD14 (Fig. 6B). Mdivi-1 treatment significantly reduced mitochondrial length and promoted mitochondrial fission (Fig. 6C).