PMC

Reverse transcription–polymerase chain reaction (RT–PCR) analysis of eight selected genes differentially expressed in invasive (T1C3) and non-invasive (IC8) clones, according to microarray analysis. (A) Semiquantitative PCR was performed with total RNA isolated from both clones. PCR products were loaded on a 1.2% agarose gel and stained with ethidium bromide. The expected length of PCR products is indicated on the right. The data are representative of three experiments. (B) Quantitative RT–PCR analysis of three genes in T1C3 cells compared with IC8 cells. Copy number was first normalised to 18SRNA levels and expressed as the fold-change over IC8 cells. The graph depicts the mean of these fold-changes ±s.d. of three independent amplifications.

Endogenous TSPAN8 silencing reduces melanoma cell invasion. T1C3 melanoma cells transfected with siRNA targeting TSPAN8 (siTSPAN8), siRNA scramble (scramble) or untransfected were seeded 2 days later into a thick layer of matrigel. (A) Micrographs depict melanoma spheroids embedded in three-dimensional matrigel immediately after seeding (day 0) and at day 3. (B) Invasive outgrowth was quantified by calculating the ratio between the area spheroid after a given incubation period to the original spheroid area at 0 h. Spheroid areas at day 0 were set to 1. Bars represent the means±s.d. of the spheroid area from quadruplicates (P<0.001). Statistical significance was assessed using Student's t-test: ^*P<0.05; ^**P<0.01; ^***P<0.001.

TSPAN8 and CAPG are strongly upregulated in T1C3 invasive melanoma cells. Invasive (T1C3) and non-invasive (IC8) melanoma cells were cell surface stained with mAbs directed against TSPAN8, RELN and FXYD5, and intracellularly stained with anti-CAPG, BCL11A, ID3 and renalase. (A) Data from a representative experiment showing flow cytometry profile of IC8 and T1C3 melanoma cells stained with the indicated mAbs. Filled histograms represent specific and open histograms isotype-matched control antibodies. (B) Results for a given protein are expressed as the following ratio: MFI in T1C3/MFI in IC8 cells ±s.d. of three independent experiments. Statistical significance was assessed using Student's t-test: ^***P<0.001. (C) Total cell lysates from IC8 and T1C3 were subjected to western blot analysis with antibodies specific for CAPG and TSPAN8, actin serving as a loading control.

TSPAN8 is expressed by invasive melanoma cells in culture and in melanoma lesions. (A) Non-invasive (M4Be, M3Ge, M3Da, M1Do cell lines and IC8, T1C11 clones) and invasive melanoma cells (WM793, SKMel28 cell lines and T1C3, TW12, T1P26 clones) were cell surface stained with antibodies directed against TSPAN8 and CAPG. Filled histograms represent specific and open histograms isotype-matched control antibodies. Results are representative of three independent experiments. (B) Representative immunohistochemical expression of TSPAN8 immunostaining in normal skin, benign nevus, RGP and VGP melanomas, and lymph node metastases. Note negative staining of cells from normal skin, except for eccrine glands, which was useful as an internal positive control. The square represents the area of magnification shown in the inset. Open arrows pointing at positive stained junctional nests of melanocytes. Black arrow pointing at a dermal nest of stained cells.

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.

Endogenous TSPAN8 silencing has no impact on cell survival, proliferation and migration. T1C3 melanoma cells were transfected with siRNA targeting TSPAN8 (siTSPAN8) or siRNA scramble (scramble). (A) Cell viability determined by XTT assay 3 days post-siRNA treatment of T1C3 melanoma cells. Data are expressed as mean OD 450 nm of siTSPAN8-treated cells/mean OD 450 nm in scramble siRNA-treated cells, ±s.d. of three independent experiments. (B) Cell proliferation determined by BrdU assay 3 days post-siRNA treatment of T1C3 melanoma cells. Data are expressed as in A. (C) Cells grown to confluence in uncoated, collagen I- or collagen IV-coated six-well plates were wounded by creating a scratch across the monolayer culture 3 days after transfection. Representative photographs showing this region were taken directly following injury (0 h) and at various time later using a Sony DXC-390 digital camera under an inverted phase microscope (Zeiss LSM510, Zeiss Inc., Thornwood, NY, USA). (D) Quantification of wound closure depicted in C. Data are expressed as percentage of the initial wound size and set to 100% at 0 h. No statistically significant difference was observed at any time point (P>0.05).