Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> (A). Typical confocal images. (i) Control. (ii) EGF (100 ng/ml). (iii) AG1478 (100 nM). Each treatment was for 24 h. Scale bar, 20 μm (applicable to all panels). (B) Signal density, ie optical density of plasma membrane (PM) VGSC immunocytochemistry, corresponding to images such as (A). Each histobar denotes mean ± standard error (n = 50 cells/3 separate experiments). (C) Effects of EGF (similar treatment as in A) on total and PM VGSC expression. The PM fraction was immunoprecipitated by biotin labelling. Key: 1) Control total VGSC protein. (2) EGF-treated total VGSC protein. (3) Control PM VGSC protein. (4) EGF-treated PM VGSC protein. Note the change in the molecular size of the biotinylated fractions (lanes 3 & 4)

Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> (A) Migration index (MiI), expressed relative to the control level (Cont), fixed as 100 %. Effects of EGF (50 ng/ml), TTX (500 nM) and EGF+TTX are shown. (B) Dose dependence of the effect of EGF on MiI. ΔMiI denotes the percentage change (increase) in MiI induced by increasing concentrations of EGF, expressed relative to the maximum (fixed as 100 %) seen for 50 ng/ml. (C) Endocytosis index (EI), expressed as percentage of the control level (Cont). Effects of EGF (20 ng/ml), TTX (500 nM) and EGF+TTX are shown. (D) Dose dependence of the effect of EGF on EI. ΔEI denotes the change (increase) in EI induced by given concentrations of EGF. (E) Boyden chamber invasion assay data. Effects of EGF (100 ng/ml), TTX (500 nM), EGF+TTX and AG1478 (100 nM) are shown. Invasion index (InvI) denotes the percentage of cells crossing the membrane in Transwell assays. Each data point or histobar denotes mean ± standard error (n = 4)

Effects of TTX (500 nM) and TTX+EGF on respective relative levels of total and plasma membrane (PM) VGSC protein expression, presented as a percentages of respective controls (Cont)

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> Two concentrations of EGF were used: 50 ng/ml (EGF1) and 100 ng/ml (EGF2). Relative levels of total VGSC were deduced from Western blots (as in Fig. 2). Relative levels of PM expression were obtained from immunocytochemistry/digital analysis(as in Fig. 4). Each histobar denotes mean ± standard error (n = 3–6). Light bars, total VGSC protein. Shaded bars, VGSC protein expressed in plasma membrane (PM)