Fig. 8

SNHG1 promotes USP8 mRNA degradation through direct interaction with HUR protein. A The SNHG1 expression level in anti-HUR pulled-down complex was analyzed using real-time PCR, with Control IgG serving as a reference. The results are depicted as relative SNHG1 levels, and bars represent the mean ± SD of triplicate measurements. An asterisk (*) indicates a significant increase compared to the complex pulled down with control IgG (p < 0.05). B A schematic diagram illustrates the binding of SNHG1 with HUR. C U5637 cells were transfected with HA-MS2 in combination with either SNHG1-MS2overexpressing or pSL-MS2-12X control constructs. Co-immunoprecipitation was executed using anti-HA antibody-conjugated agarose beads, and the immunoprecipitates were subsequently analyzed by Western blotting to detect HUR. D The USP8 mRNA expression level was assessed using qRT-PCR, with IgG acting as a control. The results are presented as relative SNHG1 levels, and bars signify the mean ± SD of triplicates. An asterisk (*) denotes a significant difference compared to the vector transfectant (p < 0.05). E HUR shRNA construct and the nonsense were stably transfected into U5637(Vector) and U5637(SNHG1); and the expression of HUR, USP8 and PTEN were evaluated by Western blot. F-G Anchorage-independent assay was performed for all cell lines as indicated, the representative images were shown (F), the number of colonies were counted and compared (G). * P < 0.05 versus U5637(Vector/Nonsense), # P < 0.05 versus U5637(SNHG1/Nonsense), Δ P < 0.05 versus U5637(Vector/shHUR). H-I Cell cycle analysis. The percentages of indicated cell line in different cell cycle stages were tested by flow cytometry, the representative results were showed (H), then they were compared by t-test (I). * P < 0.05 versus U5637(Vector/Nonsense), # P < 0.05 versus U5637(SNHG1/Nonsense), Δ P < 0.05 versus U5637(Vector/shHUR)