Fig. 1 | Oncogene

Fig. 1

From: Pathogenic mutations in neurofibromin identifies a leucine-rich domain regulating glioma cell invasiveness

Fig. 1

NF1 loss promotes GPCs invasion in vitro and in vivo. a NF1 mRNA expression in GBM subtypes. One-way ANOVA with Tukey’s test was used to analyze statistical significance, *p < 0.01. Data are represented as mean ± SD. b Neurofibromin expression in patients-derived tumor xenograft. Scale bar = 50 µm. c NF1 mRNA and protein expression in NF1-shRNA#1 and #2-transduced (i) NNI-12 and (ii) NNI-21. HSP90 serves as the loading control. d Percentage of invasion in NF1-shRNA #1 and #2-transduced NNI-12 and NNI-21. e (i) Photomicrographs show H&E staining and neurofibromin expression in shCtrl (top) and NF1-shRNA#1(bottom)-transduced tumor. Black line, invasion zone between tumor and normal; red arrows, pocket of invasive cells; T tumor, N normal. Scale bar = 25 μm. (ii) Photomicrographs show composite of seven images demonstrating the extent of invasion in NF1-shRNA-transduced tumor. Red arrow, pocket of invasive cells; black line, invasion zone betwen normal and tumor region. Scale bar = 50 μm. (iii) Quantification of the number of invaded cells in shCtrl and NF1-shRNA-transduced tumor. Data shown are absolute value per field (200× original magnification). Student’s t test was used to analyze statistical significance between NF1-shRNA and shCtrl-transduced tumor, **p < 0.001. f Immunohistochemistry staining shows Vimentin, CD44 and Sox2 expression in shCtrl (top) and NF1-shRNA (bottom) transduced tumor. Scale bar = 50 μm. g mRNA expression of (i) Vimentin, CHI3L and CD44, and (ii) Sox2 in shCtrl and NF1-shRNAs-transduced cells as determined by qPCR. h qPCR analysis of EMT markers SNAI1, ZEB2, ZEB1, and TWIST1 in shCtrl and NF1-shRNA-transduced cells. i Western blot analysis demonstrating expression of p38, STAT3, AKT, p70S6K, S6, SMAD2 in NNI-21 cells transduced with shCtrl and NF1-shRNA 72 h post-infection. Actin serves as the loading control. Densitometry quantification was done for the indicated proteins by normalizing to actin. Ratios were indicated below each blot. For (c), (d), (g), and (h), data presented are representative from three independent experiments ± SEM. Student’s unpaired t test was used to analyze statistical significance between NF1-shRNA and shCtrl-transduced cells, *p < 0.01, **p < 0.001, ***p < 0.0001

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