Fig. 5

SNHG1 attenuated USP8 expression, consequently facilitating PTEN protein degradation. A-D The selected cell extracts underwent Western blot analysis to determine the expression levels of the indicated proteins. β-Actin was used as a protein loading control. E and F UROtsa(SNHG1/Flag-USP8) and UROtsa(SNHG1/Vector) cells were evaluated for anchorage-independent growth, both with and without EGF, as specified. Following a 3-week incubation period, representative images of the colonies were captured under microscopy, and the colony count was conducted, with results presented as colonies per 50,000 cells. An asterisk (*) signifies a significant increase compared to the medium control, and the symbol (#) denotes significant inhibition relative to UROtsa(SNHG1/Vector) cells cultured in normal medium (p < 0.05), the symbol. (∆) indicates a significant decrease compared with UROtsa(SNHG1/Vector) cells treated with EGF (p < 0.05). G and H U5637 cells, including variants U5637(shSNHG1#1/shUSP8#1) and U5637(shSNHG1#1/shUSP8#2), were subjected to an anchorage-independent growth assay. The number of colonies, each containing more than 32 cells, was tallied, and results were depicted as colonies per 10⁴ cells. Bars represent the mean ± SD. I U5637 cells were transfected with Flag-USP8 or its vector control constructs, followed by coimmunoprecipitation with anti-Flag antibody-conjugated agarose beads. The immunoprecipitates were subsequently analyzed through immunoblotting to detect PTEN. J U5637 cells were transfected with ubiquitin-WT constructs in conjunction with Flag-USP8, as indicated. Transfectants were treated with the proteasome inhibitor MG132 (5 μM) for 10 h, 24 h’ post-transfection. The cells were then lysed and co-immunoprecipitated with anti-PTEN antibody-conjugated agarose beads, followed by Western blot analysis using anti-Ub to detect PTEN ubiquitination