Fig. 2 | Oncogene

Fig. 2

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

Fig. 2

NF1-LRD expression reverts cell invasion. a (i) Structure of neurofibromin. CSRD cysteine-serine rich domain, GRD GAP-related domain, LRD leucine-rich domain, CTD C-terminal domain. (ii) Vectors used in this study. The expression vector pGW1 contains the CMV promoter driving transgene expression. Transgene is tagged inframe with HA. pCDH vector is a lentivirus vector-based expression vector. Transgene expression is driven by the CMV promoter. (iii) The four domains were subcloned into pGW1 and transfected into LN229 cells. Immunoblot shows the expression of GRD, LRD, CRD, and CSRD in LN229-transfected cells 48 h post-transfection. Immunoblot was performed using antibody against HA. HSP90 serves as the loading control. b Invasion assay was performed on transfected LN229 cells. Percentage of invasion was normalized to that of the vector-transfected cells. Representative images for DAPI-positive cells were shown. n = 5 replicates. c Effect of proliferation was assessed on LN229-transfected cells at 24, 48, and 72 h post-transfection. Percentage of cell proliferation was normalized to that of vector-transfected cells at 24 h. d Invasion assay was performed on (i) U87MG, (ii) NNI-12, (iii) NNI-21 and (iv) neurofibromin-negative patient-derived GPCs. e (i) Photomicrographs show H&E staining of NF1-shRNA vector or NF1-LRD-transduced tumor. Red arrows, pocket of invasive cells; black line, invasion zone between tumor and normal region of the brain. Scale bar = 50 μm. (ii) Quantification of the number of invaded cells in NF1-shRNA vector or NF1-LRD-transduced tumor per field (200× original magnification). The number of mice with non-invasive tumor in NF1-shRNA- NF1-LRD-transduced tumor was summarized in the table. Statistical significance was analyed using Fischer’s exact test. All data are represented as mean ± SEM. Student’s unpaired t test was used to analyze statistical significance between NF1-LRD and vector, *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001

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