High filamin-C expression predicts enhanced invasiveness and poor outcome in glioblastoma multiforme

Figures

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  Fig. 1. Immunohistochemical detection of FLNC and Kaplan−Meier plot of overall survival in GBM patients. a Kaplan−Meier curves showing validation of the prognostic potential of FLNC mRNA expression in TCGA dataset (N = 153 high 84 and low 61; p = 0.0006). b, c Representative image of FLNC expression in a GBM specimen; tumours with low (b) and high (c) expression are shown. Original magnification: ×200; scale bar: 500 μm. d Kaplan−Meier curves showing the influence of FLNC expression—with median FLNC level as the cut-off point—on GBM patient outcome in terms of overall survival. Differences were estimated with the log-rank test (p = 0.0002). e, f FLNC mRNA and protein expression levels in GBM cell lines (LN229, U251MG, U87MG, and KNS81) were examined by qRT-PCR and immunoblotting, respectively. GAPDH was used as a loading control. GBM glioblastoma multiforme, TCGA The Cancer Genome Atlas, GAPDH glyceraldehyde 3-phosphate dehydrogenase

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  Fig. 2. FLNC overexpression enhanced GBM cell invasion. a FLNC mRNA and protein levels in control and FLNC-overexpressing (OE) LN229 (left) and U251MG (right) cells, as determined by qRT-PCR and immunoblotting. GAPDH was used as a loading control. b Representative images from the Transwell migration and invasion assays of FLNC OE cells. Original magnification: ×200; scale bar: 500 μm. c Quantification of control and FLNC OE cell migration and invasion. I/M indicates the invasion/migration ratio. Columns represent total cell number in five independent microscopic fields and bars indicate SD. NS not significant; *P < 0.05; **P < 0.01. GBM glioblastoma multiforme, GAPDH glyceraldehyde 3-phosphate dehydrogenase

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  Fig. 3. FLNC silencing inhibits invasiveness in GBM cell lines. a FLNC mRNA and protein levels of control and U87MG (left) and KNS81 (right) FLNC knockdown (sh) cells, as determined by qRT-PCR and immunoblotting, respectively. GAPDH was used as a loading control. b Representative images from the Transwell migration and invasion assays of control and FLNC-depleted cells. Original magnification: ×200; scale bar: 500 μm. c Quantification of control and FLNC sh cell migration and invasion. I/M indicates the invasion/migration ratio. Columns represent total cell number in five independent microscopic fields and bars indicate SD. NS not significant; **P < 0.01. GBM glioblastoma multiforme, GAPDH glyceraldehyde 3-phosphate dehydrogenase

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  Fig. 4. Effect of FLNC signalling on MMP2 activation. a MMP2 mRNA expression levels and FLNC were examined by qPCR using cell lysates. b U251MG EGFP OE cells and FLNC OE cells as well as U87MG Scramble Sh and FLNC KD cells were incubated in serum-free medium for 48 h, and the conditioned medium was analysed by gelatin zymography assay. The experiment was replicated three times. c The graph shows the numbers of U251MG EGFP, FLNC OE1, and FLNC OE2 cells that invaded through the Transwell Matrigel membrane in the presence or absence of GM6001, an MMP2 inhibitor. MMP matrix metalloproteinase