Fig. 3
The downregulation of PTEN mediated SNHG1’s promotion of urothelial cell transformation, anchorage-independent cell growth and tumorigenesis in BC. A-C The indicated cell extracts were analyzed by Western blot to determine the expression of PTEN, PHLPP1, PHPPL2, p63α, PP2A-A, PP2A-B, and PP2A-C. β-Actin served as a protein loading control. D-E AKT and p-AKT (T308) were determined by Western blot in UROtsa(SNHG1) versus UROtsa(Vector) cells (D), and T24T(shSNHG1#1) cells, T24T(shSNHG1#2) cells versus T24T(Nonsense) cells (E). β-Actin was the protein loading control. F A PTEN-overexpressing construct was stably transfected into UROtsa(SNHG1) cells, and Western blot analysis was performed to confirm PTEN protein expression. G and H UROtsa(SNHG1/PTEN) and UROtsa(SNHG1/Vector) transfectants were analyzed for anchorage-independent growth in the presence or absence of EGF, as indicated. Representative images of the colonies were captured under microscopy after a 3-week incubation period. The colony count was conducted under microscopy, with results presented as colonies per 50,000 cells. An asterisk (*) signifies a significant increase compared to the medium control, while the symbol (#) denotes significant inhibition compared to UROtsa(SNHG1/Vector) cells treated with EGF (p < 0.01). I PTEN shRNA construct was stably transfected into U5637(shSNHG1#1) cells, and PTEN knockdown efficiency was verified by Western blotting. J and K U5637(shSNHG1#1/shPTEN) cells versus U5637(shSNHG1#1/Nonsense) cells were subjected to an anchorage-independent soft agar assay. The number of colonies, each with more than 32 cells, was counted, and the results were presented as colonies/104 cells. Bars depict the mean ± SD, and an asterisk (*) indicates a significant increase compared to the U5637(shSNHG1#1/Nonsense) transfectant (p < 0.01)