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. 2015 Mar 21;6(10):8353–8365. doi: 10.18632/oncotarget.3229

Figure 3. HOTAIR regulated the activity of the β-catenin signaling pathway in vitro.

Figure 3

(A) A TOP flash reporter assay indicated that Lenti-HOTAIR si treatment inhibited β-catenin transcriptional activity (P < 0.01). (B) A TOP flash reporter assay indicated that DZENP treatment inhibited β-catenin transcriptional activity to a greater degree than 2PCPA treatment (P < 0.05). (C) Lenti-HOTAIR si treatment inhibited β-catenin and PKM2 expression and decreased p-β-catenin levels in whole cell lysates of U87 and U87vIII cells. (D) DZENP treatment inhibited the expression of β-catenin and decreased p-β-catenin levels in whole cell lysates of U87 and U87vIII cells. (E) Lenti-HOTAIR si treatment inhibited β-catenin expression and decreased p-β-catenin levels in the nucleus lysate in U87 and U87vIII cells. (F) Lenti-HOTAIR si treatment inhibited β-catenin expression and decreased p-β-catenin levels in the cytosol lysate in U87 and U87vIII cells. (G) Treatment with the Lenti-HOTAIR 5′ domain increased the levels of β-catenin and PKM2. (H) Freshly isolated cell lysates (U87 and U87vIII cells infected with Lenti-HOTAIR si or Lenti-NC) were used to immunoprecipitate β-catenin or PKM2 with specific antibodies. Whole immunoglobulin (IgG) was used as a control antibody for immunoprecipitation assays. The immunoprecipitated complexes were subjected to Western blot analysis with specific antibodies against β-catenin and PKM2 as indicated. GAPDH or Histone 2A was used as a loading control. (I) Compared with Lenti-HOTAIR si-treated cells, Lenti-NC-treated cells exhibited higher β-catenin and PKM2 expression in both the cytoplasm and the nucleus (magnification: 1000x).