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. 2019 Apr 14;16:531–542. doi: 10.1016/j.omtn.2019.04.007

Figure 2.

Figure 2

miR-146b Overexpression Was Crucial for Human Bladder Cancer Cell Invasion and Anchorage-Independent Growth

(A) miR-146b inhibitor and its nonsense control plasmids were stably transfected into T24T and UMUC3 cells, and stable transfectants were identified by real-time PCR. The error bars represent the mean ± SD. Student’s t test was used to determine the p value, and the asterisk (*) indicates a significant decrease relative to nonsense control cells (*p < 0.05). (B) miR-146a expression was dectected in the indicated cells. The error bars represent the mean ± SD. Student’s t test was used to determine the p value. (C–E) The invasion abilities of T24T (miR-146b inhibitor) (C), UMUC3 (miR-146b inhibitor) (D), and their nonsense transfectants were determined by using BD BioCoat Matrigel Invasion Chamber applied with the matrigel (E). The error bars represent mean ± SD from three independent experiments. Student’s t test was utilized to determine the p value, and the asterisk (*) indicates a significant decrease as compared with nonsense control transfectants, *p < 0.05. (F) T24T transfectants as indicated were plated in 96-well plates at a density of 5,000 cells/well. The cell culture medium was then replaced with 0.1% FBS DMEM-F12 (1:1) and cultured for 12 h. The cells were replaced with normal medium and cultured for another 1, 2, 3, or 4 days. Subsequently, ATP activity assay was performed using the protocol described in the Materials and Methods. The asterisk (*) indicates a significant decrease from nonsense control. (G) The indicated cells were subjected to anchorage-independent soft agar assay using the protocol described in the Materials and Methods. Representative images of colonies of indicated cells were photographed under an Olympus DP71. (H) The number of colonies was counted with more than 32 cells of each colony, and the results were presented as colonies per 104 cells; the error bars show mean ± SD from three independent experiments. The asterisk (*) indicates a significant decrease in comparison to those of nonsense control transfectants (**p < 0.05).