Fig 2: Expression and silencing of BLM in two bladder cancer cell lines. (A) Results of reverse transcription-quantitative PCR showing the relative mRNA expression of BLM in the invasive and well-differentiated bladder cancer cell line, 5637 and in the invasive and poorly differentiated cell line, J82. (B) Western blot analysis of the protein level of BLM in the 5637 and J82 cells. (C) BLM mRNA level in bladder cancer cells was silenced by transfection with siRNA-BLM compared with siRNA-CON-transfected cells (*P<0.0001). (D) Western blot analysis demonstrated that the BLM protein level significantly decreased in the siRNA-BLM-transfected 5637 and J82 cells compared with the siRNA-CON-transfected cells. BLM, Bloom syndrome protein; siRNA, small interfering RNA; CON, control.

Fig 3: Silencing of BLM decreases bladder cancer cell proliferation. (A) Representative results of the effect of siRNA-BLM on bladder cancer cell proliferation (magnification, ×40). (B) Quantitative analysis of EdU-positive J82 cell numbers. The numbers of EdU-positive J82 bladder cancer cells transfected with siRNA-BLM were decreased compared with the siRNA-CON group. *P<0.05. (C) Quantitative analysis of EdU-positive 5637 cell numbers. The numbers of EdU-positive 5637 cells transfected with siRNA-BLM were decreased compared with the siRNA-CON group *P<0.05. BLM, Bloom syndrome protein; siRNA, small interfering RNA; CON, control.

Fig 4: Silencing of BLM promotes bladder cancer cell apoptosis. (A) Representative results of cell apoptosis measured using flow cytometry. (B) The quantification of apoptotic cell numbers indicated that the J82 cells transfected with siRNA-BLM exhibited enhanced apoptosis compared with the siRNA-CON group. *P<0.05. (C) The quantification of apoptotic cell numbers indicated that the 5637 cells transfected with siRNA-BLM exhibited an increase in apoptosis compared with the siRNA-CON group. *P<0.05. BLM, Bloom syndrome protein; siRNA, small interfering RNA; CON, control.

Fig 5: In cellulo validation of SMYD3 interactions identified in silico. (A-E) Validation of SMYD3 interactions in HT-29 CRC cells. Co-immunoprecipitation of endogenous SMYD3 and mTOR (A), BLM (B), MET (C), p130 (D), or RPB1 (E) using specific antibodies. RPB1 was used as a control of our in silico analysis. Input corresponds to 10% of the lysate. Anti-IgGs were used as negative controls. Results are representative of at least three independent experiments. (F) P-tripeptide localization in specific domains of AMPK subunits. (G) Validation of SMYD3-AMPK interaction in gastrointestinal cancer cell lines. Co-immunoprecipitation of endogenous SMYD3 and AMPK using anti-SMYD3 and anti-AMPK antibodies in HT-29 CRC cells, HGC-27 GC cells, HLC-19 HCC cells, and CAPAN-1 PC cells. Input corresponds to 10% of the lysate. Anti-IgGs were used as negative controls. Results are representative of at least three independent experiments. (H) Validation of SMYD3 interaction with phospho-mTOR, phospho-AMPK, and phospho-MET in HT-29 CRC cells. Co-immunoprecipitation of endogenous SMYD3 and phospho-mTOR/phospho-AMPK/phospho-MET using anti-SMYD3 antibodies and antibodies against the phosphorylated form of the interacting proteins. In order to phospho-activate MET, HT-29 CRC cells were serum-starved for 24 h and subsequently treated with 10 ng/ml of HGF for 2 h. Input corresponds to 10% of the lysate. Anti-IgGs were used as negative controls. Results are representative of at least three independent experiments.