Figure 2.

CD46-CYT1 and CD46-CYT2 have opposite roles in bladder cancer development

(A) Generation of CD46-knockout (CD46-KO) cells that were engineered to re-express CD46-CYT1 or CD46-CYT2. CD46-KO cells were infected with lentiviruses expressing vector control, CD46-CYT1, or CD46-CYT2. Immunoblotting was performed to evaluate the expression of CD46. GAPDH is an internal control. (B) A CCK-8 kit was utilized to quantify cell viability at each time point. The data represent mean ± SD and were analyzed by a two-way ANOVA (n = 3). ∗∗∗p < 0.001. (C) Quantification of 5-ethynyl-2′-deoxyuridine (EdU)-incorporated cells in indicated engineered cell lines. The data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 4). ∗∗∗p < 0.001. (D) A colony formation assay and quantification were performed with EJ-1 cells and CD46-KO cells expressing CD46-CYT1 or CD46-CYT2 as described in (B). The data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 3). ∗∗∗p < 0.001. (E) Transwell cell migration assay for EJ-1 cells. Numbers of migrated cells were quantified in four random images from each treatment group. The data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (F) (1) Time course of xenograft growth. Mean tumor volume was measured by caliper on the indicated weeks. The data represent mean ± SD and were analyzed by a two-way ANOVA (n = 8). ∗∗∗p < 0.001. (2) Photographs of tumors excised 7 weeks after the inoculation of stably transfected EJ-1 cells into nude mice. (3) The tumor weight of CD46-CYT1- or CD46-CYT2-overexpressed EJ-1 cells in nude mice at the end of 7 weeks after transplantation. The data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 8). ∗∗p < 0.01.