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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Mol Cancer Ther. 2017 Feb 21;16(5):924–935. doi: 10.1158/1535-7163.MCT-16-0631

Figure 2. The differential effects of miR-145 on anchorage-independent growth between T24 and T24T cells were mediated by FOXO1.

Figure 2

(A & B), The cell extracts from T24(Vector) vs. T24(miR-145) (A) or T24T(Vector) vs. T24T(miR-145) (B) cells were subjected to Western Blotting for detection of FOXO1 and p27 protein levels as indicated. The foxo1 mRNA expression was also determined by RT-PCR as indicated. GAPDH protein or mRNA was used as loading control, respectively. (C) The expression of foxo1 mRNA was detected by Quantitative real-time PCR in 23 pairs of normal bladder (Normal) and cancerous patients (Tumor) with lymph node metastasis. (D & E), T24(Vector) and T24(miR-145) cells were stably transfected with either Nonsense shRNA or two FOXO1 shRNA constructs (shFOXO1-4, shFOXO1-6), respectively. The knockdown efficiency of FOXO1 was assessed by Western Blotting (D), and the stable transfectants were then used for determination of their anchorage-independent growth ability (E). (F & G), Flag-FOXO1 expression construct was used to stably transfected into T24T(Vector) and T24T(miR-145) cells, respectively. The overexpression efficiency was identified by Western blotting (F), and the stable transfectants were then subjected to determination of their anchorage-independent growth ability (G). Colonies with more than 32 cells were scored and presented as colonies/104 cells. Western Blotting experiments were repeated at least three times and the representative blots were shown in the figures. Results were presented as the means±SD of triplicates. Symbol “*” indicates a significant difference between Vector and miR-145 transfectants, while symbol “♣” indicates a significant difference in transfectants with either FOXO1 knockdown or overexpression in comparison to the scramble transfectant (p < 0.05).