The human lung cancer cell line A549 was cultured in RPMI 1640 medium (Gibco, Shanghai, China), and bladder cancer cell line UC3 was cultured in DMEM medium (Gibco, Shanghai, China). All cells were cultured in a 37℃ humidified incubator with 5% CO₂ and the culture medium containing 10% fetal bovine serum (FBS) (Gibco, Shanghai, China), 1% penicillin–streptomycin (Gibco, Shanghai, China), 1% L-glutamine (Gibco, Shanghai, China) and 1% non-essential amino acid (NEAA) (Gibco, Shanghai, China) in addition to the basic culture medium.

To transient knockdown BAP31 in cancer cells, siRNA 5′-CCUCCAAUGAAGCCUUUAATT-3′ and 5′-UUAAAGGCUUCAUUGGAGGTT-3′ were used this study. As a control for this study, a non-silencing negative siRNA sequence obtained from GenePharma (Shanghai, China) was used. When cells growing into 50% confluence, siRNA was transfected into cells using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) according to the manufacture’s guidelines. A final concentration of the siRNA was 100 nM. After being incubated with BAP31 siRNA for 24 hours, cells were re-transfected for another 48 hours. The knockdown cells were harvested after 72 hours.

For transient overexpression studies, a pcDNA3.1(+)-BAP31-Flag plasmid was used, and the empty vector—pcDNA3.1(+) was used as control. When the cells growing into 80% confluence, the cells were transfected using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) and harvested after 48 hours for subsequent experiments.

For BAP31 stable knockdown cell lines construction, UC3 cells were seeded into a 6-well plate at a density of 20×10⁴ cells/well. Cells were transfected with BAP31 shRNA (Novobio Scientific Co., Ltd., Shanghai, China) plasmid using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) according to the manufacture’s guidelines. After 48 hours, blasticidin was added to the medium every three days to screen successfully transfected cells. After 20 days treatment, cells were digested and seeded into 96-well plates at a density of 1 cell/well to grow into a monoclonal cell mass. Then, the expression of BAP31 gene was detected to select the positive cell clone with extremely low BAP31 expression. The positive clone cell masses were collected and stored in liquid nitrogen for subsequent experiments.

TGFβ1 was purchased from Peprotech. Chloroquine (CQ) and magnesium chloride (MgCl₂) were purchased from Sigma-Aldrich. 3-methyladenine (3-MA), 6-Bromoindirubin-3´-oxine (BIO), XAV939 and Z-VAD-FMK were purchased from Selleck. All the stock solutions were diluted in culture medium prior to experimentation. Unless otherwise specified, all other chemicals were purchased from Sigma-Aldrich.

The proliferation of UC3 BAP31 knockdown cells was detected using Methyl Thiazolyl Tetrazolium (MTT) assay, and the proliferation of BAP31 siRNA knockdown cells was examined using Cell Counting Kit-8 (CCK-8) assay. In addition, the proliferation was also further determined using soft agar cloning formation assay.

For MTT assay, UC3 BAP31 knockdown cell lines were seeded (2×10³ cells/well) into 96-well plates with five replicates. After 24 hours, 10 μl of MTT solution mixed with 100 μl culture medium was added to each well and the cells were incubated for 4 hours at 37°C. Then, the MTT solution was removed, and 100 μl of DMSO was added to dissolve the formazan crystals. Absorbance (OD) was measured using a microplate reader at 570 nm wavelength.

For CCK-8 assay, cancer cells were seeded into 96-well plates with a density 2×10³ cells/well and transfected with BAP31 siRNA or negative siRNA control. After 48 hours transfection, cells were treated another 24 hours with different concentrations of culture medium containing chemicals (TGFβ1, BIO, XAV939, MgCl₂ or Z-VAD-FMK). Finally, cells in each well were added 10 μl of CCK-8 solution mixed with 100 μl culture medium and incubated for 1.5 hours at 37°C. The absorbance of the samples was measured at a wavelength of 450 nm.

Soft agar cloning formation assay was also used to detect the viability of BAP31 knockdown cells. Briefly, 1.5 ml culture medium with 0.6% agar was used as the bottom gel and coated into 6-well plates for each well. After solidification, 1×10³ BAP31 knockdown cells were suspended in 1 ml medium with 0.35% agar and poured onto the bottom gel, and the cells were cultured for another 20 days in incubator to form colonies. In the meantime, 150 μl culture medium was added to these plates every 2 days to prevent the cells from drying. Finally, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet solution for 15 min. The stained colonies were counted and photographed for further analysis. Each group had three replicates.

Flow cytometry was used to detect the cell apoptosis and cell cycle of BAP31 knockdown or overexpress cells. In brief, 30×10⁴ cells were seeded to 6-cm dishes and transfected for 48 hours and treated with different chemicals for another 24 hours. The treated cells were incubated with propidium iodide (PI) (BD Biosciences, New York, USA) or an annexin V-FITC/PI apoptosis detection kit (Meilunbio, Dalian, China) for cell cycle and apoptosis detection according to the manufacture’s guidelines. The cells were harvested and analyzed using a flow cytometer (BD LSRFortessa, BD Biosciences, New York, USA).

Cells were washed with PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). The extracted protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Protein samples (20 μg) were separated on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After transferring, the membranes were blocked in 5% non-fat milk solution for 1 hour at room temperature, and incubated with specific primary antibodies overnight at 4°C. Then, the membranes were incubated with the appropriate HRP-conjugated secondary antibody at room temperature for 1 hour, and visualized using an ECL detection system. The sources and dilutions of the antibodies used in this study were listed in Supplementary Table S1 .

Total RNA of the BAP31 knockdown cells was isolated using TRIzol reagent (Tiangen Biotech, Beijing, China) and reverse-transcribed into cDNA using the All-in-One cDNA synthesis SuperMix kit (Bimake, Shanghai, China) according to the manufacturer’s instructions. Quantitative RT-PCR was conducted using 2×SYBR Green qPCR Master Mix (Bimake, Shanghai, China) following the manufacturer’s protocol on a CFX96 real-time PCR detection system and GAPDH was used as a reference gene. The relative expression of the target genes was calculated using the 2^-ΔΔCt method.

Transwell assay was performed by a 24-well transwell chamber (8 µm pore size, Greiner Bio-one). Treated cells (2×10⁴ cells/well) were suspended in 100 μl serum-free culture medium and seeded into the upper chamber, and 600 μl medium containing 2.5% FBS was added to the lower chamber of each well. After incubation 24 hours at 37°C, the chambers were washed thrice with PBS, fixed in methanol for 30 min, and stained with 0.1% crystal violet for 15 min at room temperature. Then, the chambers were washed with PBS and five fields were randomly selected and observed under a microscope.

Immunofluorescence assay was used to detect target protein localization and expression. Briefly, lung cancer A549 cells were seeded at 2×10⁴ cells per well into a 24-well plate and transfected with siRNA and/or TGFβ1 treatment. After that, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 30 min, and blocked in 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Then, the fixed cells were incubated with primary antibodies at 4°C overnight, rinsed thrice with 0.2% Tween-20 in PBS and incubated with the appropriate fluorescence‐conjugated secondary antibody at 4°C overnight in the dark. The sources and dilutions of the antibodies used for immunofluorescence were listed in Supplementary Table S1 . Finally, the nuclei were stained with Hoechst 33342 for 5 min. A fluorescence microscope was used to observe cells and collect pictures.

To determine β-catenin distribution in BAP31 knockdown cells, cytoplasmic and nuclear proteins were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The concentration of the extracted protein was determined by BCA protein assay kit (Beyotime, Shanghai, China) and the protein samples were detected by Western blot analysis. GAPDH and H3 were served as reference genes for cytoplasm and nucleus, respectively.

All of the experiments were performed in triplicate unless otherwise noted. The statistical analyses were conducted using SPSS 22 and GraphPad prism 6 software. The data are presented as the means ± the experimental standard error of the mean (SEM). Survival curves were generated using the Kaplan-Meier method and log-rank tests. One-way analysis of variance (ANOVA) was used to determine the statistically significant differences for multiple comparisons. Chi-square analysis was used to test the difference on proliferation or survival rate. p-value <0.05 was considered significant.

To determine the effect of BAP31 on cancers, we first investigated BAP31 expression in different types of cancer datasets [The Cancer Genome Atlas (TCGA), Taylor]. As shown in Figure 1A , the expression of BAP31 was increased in several types of cancers compared to normal tissues, including lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and bladder urothelial carcinoma (BLCA). Kaplan-Meier analyses based on patient information revealed that high BAP31 expression predicted shorter overall survivals ( Figure 1B ). The above results possible suggest that BAP31 expression is increased in lung cancer tissues and associated with poor prognosis in lung cancer patients.

Considering the potential role of BAP31 in cancer development, we firstly uncovered the impact of modifying BAP31 expression on cell proliferation. We constructed BAP31 over-expression plasmid using pcDNA3.1(+) as backbone vector. Transient expression of BAP31 over-expression plasmid was conducted in lung cancer cells A549, and CCK-8 assay was performed in the cells received transfection for 24, 48, 72 and 96 hours ( Figure S1 ). Results indicated that there was no significant difference on the absorbance value of cells among BAP31 over-expression group, pcDNA3.1(+) empty vector transfection group, and blank control group. To further determine the role of BAP31 on cell proliferation, siRNA was applied to knockdown the expression of BAP31 in A549 cells. As shown in Figure 1C , the cell proliferation was down-regulated after decreasing the expression of BAP31, especially at 96 hours. The colony formation assay was then performed in A549 cells, and results also found that the number of cell colony in BAP31 knockdown group was significantly lower than the control group ( Figure 1D ). To further confirmed that BAP31 downregulation impaired the cancer cell proliferation, we constructed two stable BAP31 knockdown cell lines using shRNA in bladder cancer cells UC3 named shBAP31-4 and shBAP31-15. Western blot analysis confirmed that the expression of BAP31 was extremely low in the BAP31 shRNA knockdown cells ( Figure S2C ). Results of MTT assay showed that the proliferation rate of BAP31 shRNA knockdown cells was lower than the control cells ( Figure S2A ). The colony formation rate in both shBAP31-4 and shBAP31-15 cells was also significantly lower than the control cells ( Figure S2B ). Generally, it seems that downregulation of BAP31 could decrease the proliferation of cancer cells.

Proliferation is closely connected with regulation on cell cycle distribution. The PI staining-based flow cytometry was used to examine the cell cycle progression in the control of BAP31. Results indicated that the proportion of cells in G0/G1 phase was increased after BAP31 knockdown by siRNA in A549 cells ( Figure 2A ). In addition, the proportion of cells in S phase was remarkedly decreased by BAP31 knockdown. To further uncover the role of BAP31 in cell cycle distribution, the expression of cyclin B1, cyclin B2, cyclin D1, CDK1, p21 and BAP31 was determined by Western blot and/or qRT-PCR ( Figures 2B, C ). Importantly, it found that the expression of BAP31 at both mRNA and protein level was decreased by siRNA. The expression of cyclin D1 and CDK1 was downregulated by BAP31 knockdown as revealed by qRT-PCR. The expression of cyclin B1 at the protein level was showed to be decreased by BAP31 knockdown. Furthermore, p21 expression was found to be upregulated by BAP31 knockdown as determined by Western blot. In sum, BAP31 knockdown induing cell cycle arrest is possibly through regulating a number of cell cycle kinases.

Cell proliferation inhibition is always accomplished with cell death activation. Apoptosis, necroptosis, and autophagy are the common cell death pathways. To determine the occurrence of cell death, annexin V-FITC/PI staining assay was performed and assessed by flow cytometry. As shown in Figure S1B , transient expression of BAP31 did not alter the viable cell proportion in lung cancer cells A549. In BAP31 knockdown A549 cells using siRNA technique, many more cells were subjected to death than the control ( Figure 2D ). Results of Western blot confirmed that the expression of BAP31 in A549 cells were decreased by siRNA ( Figure 2E ). Furthermore, the expression of pro-apoptotic protein Bax, Bak and Bid was enhanced by BAP31 knockdown, and the expression of anti-apoptotic protein Bcl2 was decreased by BAP31 knockdown, indicating that apoptosis was evoked in A549 cells after downregulation of BAP31 expression. As an important marker gene in necroptosis, MLKL expression was also promoted in the A549 cells received BAP31 siRNA. To further confirm such findings, the occurrence of cell death was also examined in bladder cancers cells. The expression of some necroptosis (RIPK1, MLKL, and PARP-1) and apoptosis (HRK, FAS, TNFRSF10A, and TNFRSF10B) related genes were accessed using qRT-PCR. Results showed that the expression of PARP-1, TNFRSF10A, and TNFRSF10B was increased in shBAP31-4 cells, and the expression of MLKL, PARP-1 and TNFRSF10A was increased in shBAP31-15 cells ( Figures S2D, E ). To further investigate the impact of BAP31 on the cell death of UC3 cells, annexin V-FITC/PI staining-based flow cytometry was applied. As shown in Figure S2F , the cell death proportion was increased after the expression of BAP31 being knockdown by siRNA. In addition, a well-known pan caspase inhibitor Z-VAD-FMK was applied to treat A549 cells in this study, and it confirmed that enhancement on cell death of BAP31 knockdown could be largely prevent by Z-VAD-FMK treatment ( Figure S3A ). Results of CCK-8 assay also demonstrated that the survival rate in BAP31 knockdown cells was reversed by Z-VAD-FMK treatment ( Figure S3B ). Our previous studies have found that MgCl₂ treatment could inhibit cancer cell survival; therefore, the present study applied MgCl₂ as a control. It found that MgCl₂ could subject more BAP31 knockdown cells into death. Generally, these results conveyed a message that downregulation of BAP31 could induce cell death in cancer cells.

Autophagy is defined as a conserved intracellular self-degradation system that is critical for cellular renovation and homeostasis in response to stress conditions. Excessive autophagy leads to a form of programmed cell death named as autophagic cell death. Therefore, the regulatory role of BAP31 in autophagy induction was investigated in A549 cells in this study. The expression of autophagy related genes was firstly examined. Results of qRT-PCR revealed that Beclin 1 and LC3 was enhanced in the cells knockdown with BAP31 siRNA ( Figure 3 ). The expression of SQSTM1/p62 was downregulated by BAP31 siRNA. However, over-expression of BAP31 in A549 cells did not largely change SQSTM1/p62 expression. In addition, BAP31 knockdown promoted the expression of LC3, and BAP31 overexpression downregulated the expression of LC3, indicating that BAP31 possibly exerted a negative role in autophagy occurrence. To further ascertain the relationship between BAP31 and autophagy, two autophagy inhibitors 3-Methyladenine (3-MA) and Chloroquine (CQ) were used to treat BAP31 knockdown cells. As shown in Figure 3B , both 3-MA and CQ could further enforce the inhibitory effect of BAP31 knockdown on cell proliferation. Furthermore, the expression of VDAC1, which is critical for mitophagy, was increased in the BAP31 knockdown cells, and its expression was decreased in the BAP31 overexpressing cells. It found that VDAC1 knockdown could also enhance the inhibitory effect of BAP31 knockdown on cell proliferation. ER stress is considered as a potent trigger for autophagy. The expression of Bip and CHOP was shown to be enhanced in the BAP31 knockdown cells at both mRNA and protein level. These results further validated that BAP31 worked as a stabilizer for autophagy induction.

Considering the high expression of BAP31 in lung cancer tissues, it urged us to determine its role in cancer cells migration and stemness. As revealed by trans-well assay, the migrated cells in BAP31 knockdown group were significantly less than the control group ( Figures 4A, B ). Expression profile of EMT related genes was then examined. Results of both qRT-PCR and Western blot analysis revealed that the expression of E-cadherin was decreased after BAP31 knockdown ( Figures 4C, E, F ). The expression of Connexin 43 was also downregulated in the BAP31 knockdown cells. The expression of N-cadherin, α-SMA and Claudin 1 was promoted in the cell knockdown by BAP31 siRNA. Thus, it seems that the EMT process was evoked in the BAP31 knockdown cells though that the migrated cells become less. Certainly, there still a sum of EMT associated genes expression indicated that BAP31 knockdown could prevent EMT process. The expression of ZO-1 and Claudin 1 was increased, and the expression of Zeb l was decreased in the BAP31 knockdown cells. To investigate the stemness of cancer cells treated with BAP31 siRNA, the expression of CD63, Sox2, TIF1β and Nanog was then analyzed. As shown in Figure 4D , the expression of CD63, Sox2 and TIF1β was upregulated by BAP31 knockdown. However, the expression of Nanog was downregulated in the BAP31 knockdown cells. We also determined the expression of cytoskeleton genes Profilin, Gelsolin, Tropomysin and Caldesmon. As shown in Figure 4G , the expression of Caldesmon was promoted in the cells treated with BAP31 siRNA. These results possibly convey a message that BAP31 participated in the migration and stemness of lung cancer cells, but its actual role should be further identified.

To exploit the actual role of BAP31 in cancer cell metastasis, the EMT process in A549 cells was induced via TGFβ treatment. As shown in Figures 5A, B , the TGFβ induced cell migration was reduced by BAP31 knockdown. However, the expression of both E-cadherin and Connexin 43 were also further downregulated in the TGFβ treated cells in combination with BAP31 knockdown ( Figure 5C ). The expression of Fibronectin, Vimentin, α-SMA and Claudin 1 in the cells with TGFβ and BAP31 siRNA treatment was higher than the cells with TGFβ treatment alone, possibly suggesting that BAP31 knockdown could activate the induction of TGFβ induced EMT process. The expression of Nanog was also further downregulated, CD63 and TIF1β was upregulated in the cells with TGFβ and BAP31 siRNA treatment ( Figure 7C ). It urged us to find out the molecular mechanism underlying BAP31 knockdown cells with potential high ability of migration but showing actual low migration proportion.

Considering the above results that BAP31 played important role in cell death, the occurrence of apoptosis in the cells received TGFβ treatment was firstly determined. As shown in Figure 6A , TGFβ treatment could also decrease the proportion of viable cells when compared with control group, and TGFβ treatment in combination with BAP31 knockdown could further enhance the proportion of death cells. Western blot analysis found that the ratio of Bax/Bcl2 was enhanced in the cells with TGFβ and BAP31 siRNA treatment ( Figure 7A ). The expression of MLKL in the cells with combinatorial treatment of TGFβ and BAP31 siRNA was also highest among the examined groups. In addition, the expression of SQSTM1/p62 and Beclin 1 was decreased, and the expression of LC3 was increased in the cells with TGFβ and BAP31 siRNA treatment when compared to the control. These results demonstrate that downregulation of BAP31 would make TGFβ treated cancer cells be in favor of undergoing apoptosis, necroptosis and autophagy.

The cell cycle distribution was examined by flow cytometry using PI staining. As shown in Figure 6B , TGFβ treatment was shown to decrease the proportion of cancer cells at G0/G1 phase. Furthermore, BAP31 knockdown could make more TGFβ treated cells into G0/G1 phase. Results of CCK-8 assay also found that BAP31 knockdown could downregulate the proliferation of TGFβ treated cells ( Figures 6C, D ). Compared to the cells with TGFβ treatment alone, the expression of p21 and cyclin B1 was shown to be decreased in the cells with both TGFβ and BAP31 siRNA treatment ( Figure 7B ), indicating that BAP31 participated in the regulation of cell cycle in TGFβ induced EMT process.

The AKT, ERK and Wnt signaling are all important pathways for cell survival. As shown in Figure 7D , the phosphorylation of AKT at both Threonine 308 and Serine 473 was decreased in the cells with both TGFβ and BAP31 siRNA treatment. The phosphorylation of p38 MAPK was found to be upregulated in the TGFβ treated cells in combination with BAP31 siRNA treatment. The expression of both Wnt5A/B and β-catenin was decreased in the cells with both TGFβ and BAP31 siRNA treatment. To further uncover the role of these multiple signaling pathways in BAP31 participated cell death induction in cancer cells, Wnt signaling was selected to be further investigated. As shown in Figure 8A , the expression of both Wnt5A/B and β-catenin was also decreased in the nuclear protein from cells treated with both TGFβ and BAP31 siRNA. Meanwhile, results of immunofluorescence also confirmed that BAP31 knockdown would prevent the β-catenin translocate into nuclear ( Figure 8C ). Therefore, it urged us to consider whether activation of Wnt signaling would reverse the cell death induction of BAP31 knockdown. As shown in Figure 8B , BIO treatment could promote the survival rate in BAP31 knockdown A549 cells. In contrast, applying Wnt signaling inhibitor XAV939 could further decrease the proliferation in BAP31 knockdown cells. Results of cytometry analysis also found that BIO treatment could prevent the death in cells transfected with BAP31 siRNA ( Figure 8D ).