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. 2015 May 19;6(24):20145–20159. doi: 10.18632/oncotarget.3980

Figure 3. NLK negatively regulates Wnt signaling pathway and its downstream targets.

Figure 3

A. Mock and NLK-WT cells have been stained with PI and cell cycle was examined by flow cytometry. P-values were calculated using the Fisher's exact test (p < 0.01). B. Immunoblots of Caspase 3 in GBM cells transduced with Mock or NLK-WT. C. Immunoblots of Wnt related proteins; anti-Flag, NLK, LEF1, Cyclin D1, and c-Myc. D. The luciferase reporter assay was used to study TCF/LEF promoter activity in Mock or NLK-WT GBM cells. (+ SD, n = 3). E. Flow cytometric analysis of GFP and apoptosis in NLK-WT GBM Cells. GBM cells were cultured and analyzed after 1, 3, 5, or 7 days post NLK-WT transduction. Bar graph represents relative GFP positive and apoptotic cell percentage in the given days. F. Real-time RT-PCR of NLK effects on mRNA expression levels of beta-catenin and its downstream target genes (MYC, FOSl1, DKK1, JUN, BTRC, and PITX2). G. Immunoblots of Wnt related proteins in mock, NLK-WT, and NLK K/N GBM Cells; anti-Flag, NLK, phosphor-LEF1, LEF1 and Cyclin D1. H. Relative luciferase activities of mock, NLK-WT, and NLK K/N GBM cells I. Immunoblots of NLK, LEF1 and phosphor-LEF1 in GBM cells transduced with mock, NLK-WT, LEF1, LEF1-2A mutant, NLK-WT/LEF1, or NLK-WT/LEF1-2A mutant. J. Comparison on the effects of mock, NLK, LEF1-2A mutant and NLK/LEF1-2A mutant on in vitro proliferation. (+SD, n = 5) K. Limiting dilution assays (LDA) for in vitro tumor sphere formation. L. Relative Luciferase activities of Mock, NLK-WT, LEF1–2A, and NLK/LEF1–2A GBM cells. M. Real-time PCR analysis of beta-catenin and its downstream target genes.