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. 2010 Nov 16;104(1):155–165. doi: 10.1038/sj.bjc.6605994

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Copyright © 2011 Cancer Research UK

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Knocking down of endogenous TSPAN8 expression is efficient and specific. T1C3 melanoma cells were transfected with either TSPAN8-specific (siT) or control scramble siRNAs (Sc). (A) Total RNA was extracted at the indicated time after transfection and transcript levels of TSPAN8 were detected by qRT–PCR. Relative mRNA expression of TSPAN8 in different cell lines was normalised to the signal intensity of 18S RNA as an internal control. A representative experiment of three independent transfection assays is shown. (B) Cell lysates from siRNA-treated cells were subjected to western blot analysis with antibodies specific for TSPAN8 or actin at the indicated days after transfection. (C) Melanoma cells were cell surface stained using the indicated mAbs at day 3 after transfection. Data from a representative experiment showing flow cytometry profile of cells transfected with scramble (black) or TSPAN8 (white) siRNAs. Mean fluorescence intensity is reported in each histogram. (D) Results for a given tetraspanin are expressed as MFI of T1C3 transfected with scramble siRNA/MFI of T1C3 transfected with TSPAN8 siRNA, and are the mean±s.d. of six independent experiments. Statistical significance was assessed using Student's t-test: ^***P<0.001.