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. 2018 Sep 9;8(17):4805–4823. doi: 10.7150/thno.25025

Figure 2.

Figure 2

WNT6 promotes GBM aggressiveness in vitro. (A-B) The efficiency of WNT6 silencing in U373 and SNB19 glioblastoma cells was analyzed by qRT-PCR and WB (A), and immunofluorescence (B). (A) Top: WNT6 expression levels were normalized to TBP. Bottom: WB images are representative of 3 independent assays; α-tubulin was used as reference protein. (B) DAPI was used to stain the nucleus (40x magnification; scale bar = 50 µm). (C-D) Cell viability was measured by trypan blue (C) and MTT (D) assays in shCtrl and shWNT6 cells. (E) Cell proliferation was evaluated by BrdU incorporation. (F-G) Matrigel invasion assays were used to assess the cells' invasion capacity. Representative images (F) with cell nuclei stained with DAPI (scale bar = 250 µm) and quantification (G) of invasive U373 and SNB19 shCtrl and shWNT6 cells. (H-K) Migration (wound healing) assays were performed in U373 (H-I) and SNB19 (J-K) shCtrl and shWNT6 cells to assess the cells' migration capacity. Representative micrographs throughout time for U373 (H) and SNB19 (J) cells (40x magnification; scale bar = 250 µm). The wound gap was measured and expressed as a percentage of wound closure for U373 (I) and SNB19 (K) cells (**** p <0.001; two-way ANOVA post-hoc Sidak's test). Results represent data from at least 3 independent experiments (mean ± SD). *, p < 0.05; **, p < 0.01; ***, p < 0.005 and ****, p < 0.001 (unless otherwise stated, a two-sided unpaired t-test with Welch's correction was applied when homoscedasticity was not verified).