(A) Immunoblot analysis to assess levels of TNC in human melanocytes and in melanoma cell lines. Increased levels of TNC in multiple melanoma cell lines as compared to normal melanocytes. Foreskin melanocytes: Fom 104 and Fom 105; melanoma cell lines: WM35, WM115, WM793, WM164, WM278, 1205Lu, and 451Lu. (B) TNC expression in either sphere-forming melanoma cells or adherent melanoma cells. Increased levels of TNC in sphere-forming melanoma cells (denoted with a –F suffix) as compared to adherent melanoma cells. Melanoma cell lines: WM35, WM3734, WM3248 and WM115; fibroblasts: FF2558. β-actin immunoblotting was performed as a loading control. The furthermost left lane was originally the last lane; the location was switched for clearer comparison of WM35-F and WM35.

(A) Immunoblot of WM3734 cells infected with TNC lentiviral shRNAs to two target sequences (sh_TNC_A and sh_TNC_C). A non-targeted shRNA (sh_Cont) was used as control. β-actin indicated equal loading. (B) Growth of WM3734 cells transduced with TNC shRNA as adherent cultures (left) and as spheres (right) was measured by MTS assay. OD 490 of each time point was normalized to Day 1. Data represent the mean ± SD (n=4). *, p<0.05 when compared to sh_Cont cells.

(A) Representative lungs showing decreased metastasis in mice injected with TNC-shRNA-expressing (sh_TNC_A or sh_TNC_C) versus control (sh_Cont) cells. 2×10⁵ WM3734 melanoma cells were injected intravenously into NOD/SCID IL2Rγ^null mice. Mice were sacrificed after 8 weeks and the lungs harvested. (B) Representative lungs stained with H&E. Scale bar = 5 mm (C) The percentage of total area occupied by lung tumor metastases was measured based on H&E stained sections. Data are means ± SE (n=8). *p< 0.05, Student’s t test. (D) WM3734 melanoma cells at a concentration of 5 × 10⁵ cells/ mouse in 0.1mL Matrigel® were subcutaneously injected into NOD/SCID IL2Rγ^null mice (n=8). Mice were monitored and the tumor size was measured twice per week.

(A) WM35, WM3734, and WM115 sphere-forming cells were stained with Hoechst 33342 dye and propidium iodide in the presence or absence of verapamil and analyzed by flow cytometry. After excluding propidium iodide-positive cells (dead cells), Hoechst dye stained cells were detected by a UV laser. When dye fluorescence was triggered at red and blue emission wavelengths, a gradient of Hoechst intensity was seen as a comma-like region on the bottom-left portion of the X-Y plot. The tip of the comma-like region included cells that efficiently excluded the Hoechst dye, therefore showing less intensity. Incubating cells with verapamil from the beginning of the Hoechst dye exposure blocked transporters that efflux the dye, and the cells expressing verapamil-sensitive transporters became Hoechst dye-positive. The lines were drawn to gate the “side population (denoted as SP)” with verapamil-sensitive transporters, which formed the tip of the comma-like region in the absence of verapamil and disappeared in the presence of verapamil. The numbers in the plots indicate percentage of side population gate among live cells. (B) Hoechst dye exclusion assay revealed that WM3734 melanoma sphere cells contained a larger SP fraction than WM3734 adherent cells. (C-D) WM3734 spheres sorted by flow cytometry for SP and major population (MP) cells. (C) qRT-PCR shows higher expression levels of ABCG2 in WM3734 MP cells compared to the SP. Data were normalized to GAPDH. The data are presented as means ± SE. (n=4) p<0.05 when compared to MP cells. (D) qRT-PCR shows higher expression levels of ABCB5 in WM3734 SP cells compared to the MP. Data were normalized to GAPDH. Data are expressed as means ± SE. (n=4) p<0.05 when compared to MP cells. (E) qRT-PCR shows higher expression levels of ABCB5 in WM3734 cells compared to WM115 cells. Data were normalized to GAPDH. Data are expressed as means ± SE. (n=4) *, p<0.05 when compared to WM3734 cells. (F) Dose response curves showing a shift in the SP compared to the MP, suggesting SP cells are more resistant to doxorubicin. Following cell sorting SP and MP cells, in single cell suspension, were plated in 96-well plates at a concentration of 10,000 cells/well in hESCM4 medium. 24 hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to MP cells.

(A) Hoechst dye exclusion assay showed that the percentage of SP cells was dramatically decreased in TNC lentiviral shRNA infected WM3734 (upper panels) and WM35 cells (lower panels). Lower right panel – immunoblot showing efficacy of TNC knockdown (sh_TNC_A) in WM35 cells compared to non-targeting shRNA (sh_Cont). β-actin indicates equal loading. (B) Gene expression of ABCB5, ABCB4 and ABCA1 was down-regulated in TNC lentiviral shRNA transfected cells. qRT-PCR analysis was performed using transporter-specific primers. Data were normalized to GAPDH and expressed as means ± SE. (n=4) p<0.05 when compared to sh_Cont cells. (C) Immunoblot showing that protein expression of ABCB5 is decreased in WM3734 cells transduced with TNC shRNA (sh_TNC_A and sh_TNC_C) when compared to non-targeting shRNA (sh_Cont). β-actin indicates equal loading. (D) Gene expression of ABCB5 was up-regulated in WM115 melanoma spheres (left) and WM3734 adherent cells (right) treated with 10 μg/ml human TNC for 24 hours. Cont: control cells cultured in regular medium. Gelatin: cells cultured in presence of 10 μg/ml gelatin matrix. qRT-PCR analysis was performed using ABCB5-specific primers. Data were normalized to GAPDH and expressed as means ± SE. (n=4) p<0.05 when compared to Cont cells.

(A) Depletion of TNC with shRNA sensitizes melanoma cells to doxorubicin treatment, demonstrated as a shift of the sh_TNC dose response curves compared to sh_Cont cells. sh_TNC and sh_Cont cells were dissociated into single cell-suspensions in hESCM4 medium, which was conditioned for 7 days with each lentiviral infected cell line, and plated in 96-well plates at a concentration of 10,000 cells/well. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to sh_Cont cells. (B) Apoptosis assessed by propidium iodide (PI) staining of untreated and 2μM doxorubicin-treated WM3734 sh_Cont cells and sh_TNC_A cells 24 hours post-treatment. Columns indicate propidium iodide-positive apoptotic cells. Twenty-four hour treatment with 2μM doxorubicin induced apoptosis in sh_TNC_A cells but did not impair survival of sh_Cont cells. (n = 3) *, p<0.05 when compared to untreated cells. (C) Exogenous TNC increases doxorubicin resistance, demonstrated as a shift in the dose response curves. WM115 sphere cells were dissociated into single cell-suspensions in hES medium and plated in 96-well plates at a concentration of 10,000 cells/well. Cont: without additional reagent. Gelatin: with 10 μg/ml gelatin matrix. TNC: with 10 μg/ml human TNC. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to Cont cells.