XMD8-92 was purchased from Tocris Bioscience (Bristol, UK). Primary antibodies, phosphorylated (p)-ERK5 (cat. no. 3371S), p-c-Jun (cat. no. 9164S), p-c-Fos (cat. no. 5348), E-cadherin (cat. no. 3195S), N-cadherin (cat. no. 4061) and Vimentin (cat. no. 3932S) were all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies for zonula occludens (ZO)-1 (cat. no. sc-8146) and GAPDH (cat. no. sc-20357) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Primers for E-cadherin, ZO-1, N-cadherin, Vimentin and GAPDH were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Male BALB/c mice (weight, 18–22 g; age, 8 weeks; n=12) were purchased from the Animal Research Center of Nanjing Medical University (Nanjing, China). Mice were group-housed in polypropylene cages, maintained on a 12 h light/dark cycle, at 22±0.5°C room temperature, at 40–60% humidity with free access to water and the AIN-76A diet. Animals were handled in accordance with the recommendations in the guidelines of the Animal Care and Welfare Committee of Nanjing Medical University. The present study protocol was approved by the Committee of the Ethics of Animal Experiments of Nanjing Medical University.

The control group (n=6) was exposed to filtered air (FA), and the TS-exposure group (n=6) was exposed to TS in a smoking apparatus designed by the authors. One commercial cigarette (Hongtashan, one of the most consumed cigarettes in China, which contains 12 mg tar and 1.1 mg nicotine per cigarette) was combusted to generate TS by a smoke machine at a constant rate (each cigarette took ~5 min to burn out). The smoke was delivered to whole-body exposure chambers with a target concentration of total particulate matter (TPM) of 80 mg/m³. Mice were exposed for 6 h daily for 12 weeks. Exposures were monitored and characterized as follows: For the control group, carbon monoxide was at 13.98±2.65 mg/m³ and TPM was at 0 mg/m³; and for the TS-exposure group, carbon monoxide was at 168.77±19.36 mg/m³ and TPM was at 81.05±3.82 mg/m³. Following the final TS exposure, mice were sacrificed by exposure to 20% CO₂ and the bladder tissues were isolated, frozen and stored at −80°C until analysis.

A total of 24 mice were divided into four groups (n=6/group) as follows: i) FA-exposure group, in which mice were exposed to FA; ii) TS-exposure group, in which mice were exposed to TS; iii) TS + dimethyl sulfoxide (DMSO) group, in which mice were injected with 15 µl DMSO and exposed to TS; and iv) TS + XMD8-92, in which mice were injected with XMD8-92 and exposed to TS. XMD8-92 was reconstituted in DMSO and injected intraperitoneally (2 mg/kg body weight) every other day. Mice were weighed weekly. Following the completion of exposure, mice were sacrificed and bladder tissues were collected for analysis.

Proteins were extracted from bladder tissues (~200 mg) using a lysate buffer (5 mmol/l EDTA, 50 mmol/l Tris, 1% SDS, pH 7.5, 10 µg/ml aprotinin, 1% sodium deoxycholate, 1% NP-40, 1 mM PMSF, 1% Triton-X 100, and 10 µg/ml leupeptin). Protein concentrations were quantified using a BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (60 µg/lane) was loaded on 10% SDS-PAGE and then transferred to polyvinylidene membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked by 5% (w/v) non-fat milk for 1 h at room temperature and subsequently probed with primary antibodies p-ERK5 (1:500), p-c-Jun (1:500), p-c-Fos (1:500), E-cadherin (1:1,000), N-cadherin (1:500), Vimentin (1:1,000) and ZO-1 (1:500), GAPDH (1:5,000) overnight at 4°C, and then incubated with a horseradish peroxidase-conjugated goat anti-mouse (1:5,000) and goat anti-rabbit (1:10,000) secondary antibodies. Immunoreactive bands were detected by enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK).

RNA was isolated from frozen bladder tissues (~100 mg) using the RNAiso Plus kit according to the manufacturer's protocol (Takara Bio, Inc., Otsu, Japan). RNA (2 µg) was reverse transcribed into cDNA using AMV Reverse Transcriptase (Promega Corporation, Madison, WI, USA). cDNA was analyzed by PCR using the Power SYBR-Green Master Mix (Takara Bio, Inc.). The thermal cycling profile for PCR was 94°C for 5 min, followed by 36 cycles of 30 sec at 94°C, with 30 sec annealing intervals at their correct temperatures (56–60°C) and 30 sec at 72°C. The primers used were as follows: E-cadherin, forward 5′-TCGACACCCGATTCAAAGTGG-3′, reverse 5′-TTCCAGAAACGGAGGCCTGAT-3′; ZO-1, forward 5′-GCAGCCACAACCAATTCATAG-3′, reverse 5′-GCAGACGATGTTCATAGTTTC-3′; Vimentin, forward 5′-CCTTGACATTGAGATTGCCA-3′, reverse 5′-GTATCAACCAGAGGGAGTGA-3′; N-cadherin, forward 5′-ATCAAGTGCCATTAGCCAAG-3′, reverse 5′-CTGAGCAGTGAATGTTGTCA-3′; and GAPDH, forward 5′-GCTGCCCAACGCACCGAATA-3′, reverse 5′-GAGTCAACGGATTTGGTCGT-3′; GAPDH served as a control. Fold alterations in gene expression were calculated by a comparative threshold cycle (Cq) method using the formula 2^−ΔΔCq (27,28).

Data are expressed as the mean ± standard deviation. Statistical analyses were performed with SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used for comparison of statistical differences of multiple groups, followed by the least significant difference test. An unpaired Student's t-test was used for the comparison between two groups. P<0.05 was considered to indicate a statistically significant difference.

TS is one of the key risk factors for BC, and TS-induced EMT is important in TS-associated malignant transformation. The present study investigated whether TS exposure induces EMT in bladder tissues. BALB/c mice were exposed to TS for 12 weeks, and the expression of the epithelial and mesenchymal markers in the bladders of mice were examined. RT-qPCR results revealed that TS exposure decreased the mRNA expression levels of E-cadherin and ZO-1. Conversely, the mRNA expression levels of Vimentin and N-cadherin were increased (P<0.01 vs. FA control; Fig. 1A). TS exposure reduced E-cadherin and ZO-1 protein expression levels, and elevated Vimentin and N-cadherin protein levels, as demonstrated by western blot analysis (Fig. 1B).

To determine if TS-induced bladder EMT alterations are associated with ERK5 activation, the expression levels of p-ERK5 were measured. It was demonstrated that TS exposure activated the urocystic ERK5 pathway (Fig. 2A). TS exposure also increased AP-1 protein expression in the bladder of mice, as indicated by elevated levels of p-c-Jun and p-c-Fos (Fig. 2B). The data therefore suggested that ERK5 activity may be important in TS-elicited EMT in bladder tissue.

The aforementioned results revealed that TS-induced urocystic EMT was associated with ERK5 activation; therefore, the present study aimed to further determine the role of ERK5 in urocystic EMT regulation. Mice were treated with XMD8-92 (2 mg/kg body weight), a highly specific ERK5 inhibitor that suppresses ERK5 activation. Western blot analyses revealed that XMD8-92 downregulated p-ERK5 expression levels (Fig. 3A). In addition, treatment with XMD8-92 markedly decreased TS-induced AP-1 activation (Fig. 3B).

To further determine the role of ERK5 in TS-triggered EMT in the bladder tissue of mice, the expression of the EMT markers were examined. ERK5 suppression reversed TS-induced alterations in the mRNA levels of E-cadherin, ZO-1, Vimentin (P<0.01 vs. FA control; P<0.001 vs. TS) and N-cadherin (P<0.01 vs. FA control; P<0.05 vs. TS; Fig. 4A). Western blot analyses demonstrated that XMD8-92 treatment attenuated both the TS-induced decrease of E-cadherin and ZO-1 levels, and the increase of Vimentin and N-cadherin in the bladders of the mice (Fig. 4B). These data demonstrated the regulatory role of ERK5 in TS-induced EMT in the bladder tissue.