In fact, EPO-R-positive cells formed melanoma lesions in NSG mice within 6C19 weeks, and these melanoma lesions were found to contain again melanoma-initiating cells when injected into secondary recipient mice. qPCR analysis using primers specific for cytokine receptor genes, additional target genes, and a set of control genes (demonstrated in Table S1c in File S1). qPCR was performed as explained in the text. Results display the percent of mRNA relative to control (set of house keeping genes?=?HKGs) and represents the meanS.D. from three self-employed experiments. All other mRNA species tested were not indicated in the melanoma cell lines examined: G-CSF-R, ABCG2, KIT, CD20, AC133/CD133, FLT-3/CD135.(TIF) pone.0084417.s002.tif (1.8M) GUID:?EA008FA8-B086-4F22-A2C2-F486BCD40750 Figure S3: Growth-promoting effect of stem cell element (SCF) in pores and skin melanocytes. cell death fluorescein detection kit (Roche, Mannheim, Germany) following a manufacturer’s protocol. Briefly, slides were fixed with 4% paraformaldehyde (60 moments), washed, and permeabilized with permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate) at 4C for 5 minutes. Then, slides were washed and incubated with 30 l TUNEL reagent at 37C for 60 moments. After washing, nuclei were counter-stained with TO-PRO-3 (100 nM) (Invitrogen) for 5 minutes. Then, slides were washed and mounted using Vectashield mounting medium (Vector Laboratories). Slides were analyzed by a fluorescent laser scanning (LSM510) confocal microscope Axiovert 200M (Carl Zeiss, Jena, Germany) using Goal version 4.2 software (Carl Zeiss). Statistical analysis To determine the significance in variations in growth and percentage of apoptotic cells the student’s t test for dependent samples was applied. Results were regarded as statistically significant when p was 0.05. Results Phenotypic definition of melanoma cells in main tumor samples Patient-derived melanoma cells were defined as CD45?/CD31? cells co-expressing CD146 and/or CD166 and/or CD63. In most cases, ANGPT1 all three markers were found to be indicated on melanoma cells, and there were no major variations in manifestation of melanoma markers when comparing freshly isolated, passaged, or xenograft-derived cells. Melanoma cell lines were found to stain uniformly positive for CD146 and CD166. The percentage of CD63+ cells in melanoma cell lines was 39% in 607B cells, 42% in A375 cells, 87% in SK-Mel28 cells, and 89% in Mel-Juso cells. Normal skin melanocytes were also found to express CD146 ( 95% cells), CD166 ( 95% cells) and CD63 (30C40% cells reactive). Manifestation of cytokine receptors on melanoma cells A number of cytokine receptors were found to be expressed on Anemarsaponin E human being melanoma cells. The Anemarsaponin E ErbB3 receptor was indicated on freshly isolated melanoma cells in all samples tested, and in most samples of cultured or xenograft-derived melanoma cells (Table 2, Table S1d in File S1 and Number 1A). The IGF-1-R was indicated on melanoma cells in 6 of 15 patient-derived melanoma samples tested, namely in 3 samples of freshly isolated cells and in 3 xenograft-samples (Table 2, Table S1d in File S1 and Number 1A). The EPO-R was found to be indicated on freshly isolated melanoma cells in all patients examined (Table 2, Table S1d in File S1, Number 1B). However, the levels of EPO-R on melanoma cells assorted from patient to patient (range: 4% to 40%) (Table S1d in File S1). In contrast to additional antigens, the EPO-R was found to be indicated on Anemarsaponin E a distinct subpopulation of melanoma cells co-expressing CD24 (Number 1B). Expression of the EPO-R on melanoma cells could also be confirmed by using directly labeled (biotinylated) rh EPO (Number 1C). Another cytokine receptor detectable in a distinct subpopulation of melanoma cells was ErbB4 (Table 2, Table S1d in File S1, Number 1D). Endoglin (CD105), a component of the TGF?1-R complex, was expressed about cultured and xenotransplant-derived melanoma cells in all individuals (range: 32C63%), whereas in freshly isolated melanoma cells, CD105 was only expressed at.