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. 2011 Jan 14;433(Pt 3):515–525. doi: 10.1042/BJ20101562

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© 2011 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(A) The expression of CIC mRNA in SKMEL13 cells treated with CIC siRNAs (siCIC, black bars) compared with mock-transfected controls (grey bars). Values are means±S.D. (four different sets of analyses for each data point). (B) The expression of ETV1, ETV4 and ETV5 mRNA in the same SKMEL13 cells treated with CIC siRNAs that are shown in (A), with data for siRNA-treated cells in black and mock-transfected controls in grey. Values are means±S.D. (four different sets of analysis for each data point). (C) The effect of CIC knockdown followed by exposure to 2 μM PD184352 on ETV1, ETV4 and ETV5 mRNA expression in SKMEL13 cells. Black bars represent data for cells treated with siCIC (siRNA for CIC) and PD184352 for the times indicated, whereas grey bars are for mock-transfected cells treated with PD184352. Values are means±S.D. (four different sets of analyses for each data point). (D) Cells at 30–40% confluency in six-well plates were transfected with siCIC, non-targeting siRNA that does not target any known genes as a control (siNT) and mock transfection. After 24 h, cells were serum-deprived for 2 h, detached with cell dissociation buffer (Gibco), counted, and added in migration buffer (750 μl of DMEM containing 1% BSA) to the upper chambers of a growth factor-reduced Matrigel™ invasion device (BD Biosciences) with chemoattractant (DMEM containing 10% FBS) in the lower wells. The cells that did not migrate were removed from the upper face of the filters using cotton swabs, and cells that had migrated to the lower face of the filters were fixed with Reastain Quick-Diff kit (Reagena). Images (×10) were captured (examples are shown in Supplementary Figure S2 at http://www.BiochemJ.org/bj/433/bj4330515add.htm), migrated cells counted in five random fields, and the histogram shows the number of cells that migrated to the lower face of the filters, presented as means±S.D. for the two experimental conditions (that is experiments starting with 2.5×10⁵ and 1×10⁵ cells respectively).