Research Paper Volume 12, Issue 3 pp 2030—2048

High expression of SMYD3 indicates poor survival outcome and promotes tumour progression through an IGF-1R/AKT/E2F-1 positive feedback loop in bladder cancer

Figure 2. SMYD3 is required for tumorigenecity of T24 and 5637 cell lines. (A) A CCK-8 assay was performed to measure the growth rate of T24 and 5637 cells at 24, 48 and 72 h post SMYD3 siRNA treatment or shRNA vector transfection. Three independent transfections were performed in triplicate. Two-way ANOVA test were used to calculate the two-sided P values. ** P < 0.01. (B) Western blot analysis of SMYD3 protein expression in T24 and 5637 cells transfected with SMYD3 siRNA for 72 h (n=3). (C) Western blot analysis of SMYD3 expression in BC cells stably transfected with the SMYD3 shRNA vector #1, #2 or control vector. GAPDH served as a loading control (n=3). (D) Representative images of clonogenic assays of the T24 and 5637 cell lines stably expressing SMYD3 shRNA #1 and #2 or control shRNA. Briefly, 200 cells/well (in 6-well plates) were incubated for 14 days (n=6). (E) Quantification of clonogenic assays for 6 independent transfections. Wilcoxon signed-rank tests for paired samples were used to calculate the two-sided P values. (FM) Xenograft model of BC in nude mice. T24 (FI) and 5637 (JM) Cells stably expressing SMYD3 shRNA or control shRNA were injected subcutaneously into BALB/c nude mice in the inguinal area (n = 8), and tumor sizes, weights and morphology were evaluated 6 weeks after injection. (F, J) Representative nude mice injected with BC cells expressing SMYD3-shRNA (blue arrow) or Control shRNA (red arrow). (G, K) Representative tumors derived from BC cell-injected nude mice. (H, L) Tumor weights of BC cells expressing SMYD3-shRNA or con-shRNA (t-test). (I, M) IHC of tumor sections from cell-injected nude-mice using SMYD3 antibody; representative staining is shown. Bars: standard deviations (SD).