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SC_12-0080_sm_supplFigure1.pdf877KSupplemental Figure 1. S100, MelanA and Vimentin immunolocalization in patient-derived shortterm melanoma cultures. Patient-derived adherent melanoma cells were fixed with 4% PFA, blocked with 10% goat serum in 0.1% Triton X in PBS and incubated with rabbit anti-S100 (Dako), mouse anti-MelanA/MART-1 (clone A103, Dako) and mouse anti-Vimentin (clone V9, Santa Cruz). Secondary antibodies were anti-mouse Rhodamine Red-conjugated and anti-rabbit FITCconjugated (Invitrogen). (Scale bar = 15 μm).
SC_12-0080_sm_supplFigure2.pdf168KSupplemental Figure 2. qRT-PCR analysis of PTCH1, SMO, GLI1, GLI2, NANOG, SOX2, KLF4, OCT4 and MITF-M expression in A375 cells (white bar) and in 14 patient-derived short-term melanoma cultures, 1 of which from a primary melanoma (black bar) and 13 from metastatic melanomas (grey bars). The y-axis in each graph represents expression ratio of gene/(GAPDH+ βACTIN average). Error bars indicate SEM.
SC_12-0080_sm_supplFigure3.pdf96KSupplemental Figure 3. Tumorigenic potential of melanomaspheres. Growth curves of melanoma xenografts, generated by injection of different doses of melanomaspheres-derived M5 and M26c cells (n=8 for each group). Six- to 8-week-old female athymic-nude mice (Foxn1 nu/nu) (Harlan Laboratories) were injected s.c. in lateral flanks with 103, 104 and 105 cells from M5 and M26c spheres. Cells were resuspended in Matrigel (BD Biosciences)/DMEM (1/1) before inoculation. Animals were monitored daily, subcutaneous tumor size was measured every 2-3 days by a caliper and tumor volumes were calculated using the formula V=W2xLx0.5, where W and L are tumor width and length, respectively.
SC_12-0080_sm_supplFigure4.pdf403KSupplemental Figure 4. Tumorigenicity of ALDHhigh and ALDHlow melanoma cell line A375. A) Tumor growth curves were generated from 103 FACS-sorted ALDHhigh and ALDHlow A375 cells (n=12 for each group). B) Representative images of A. Six- to 8-week-old female athymic-nude mice (Foxn1 nu/nu) (Harlan Laboratories) were injected s.c. in lateral flanks with 103 FACS-sorted ALDHhigh and ALDHlow cells from A375 spheres. Cells were resuspended in Matrigel (BD Biosciences)/DMEM (1/1) before inoculation. Animals were monitored daily, subcutaneous tumor size was measured every 2-3 days by a caliper and tumor volumes were calculated using the formula V=W2xLx0.5, where W and L are tumor width and length, respectively. Error bars represent SEM (Scale bar = 10 mm).
SC_12-0080_sm_supplFigure5.pdf1100KSupplemental Figure 5. Immunocytochemistry for SMO (upper panels) and in situ hybridization for GLI1 mRNA (lower panels) in tumors derived from subcutaneous injection into athymic nude mice of 1000 ALDHlow (left panels) and ALDHhigh (right panels) SSM2c cells. Formalin-fixed paraffin-embedded sections were subjected to immunohistochemical staining with rabbit antihuman SMO (Abcam) antibody followed by incubation with a secondary antibody and visualization by using UltraVision Detection System (Lab Vision, Fremont, CA) and DAB (Dako, Carpinteria, CA) for SMO, according to manufacturer's recommendations. In situ hybridization with digoxygeninlabelled antisense RNA probes for GLI1 was as previously described [26]. (Scale bar = 120 μm).
SC_12-0080_sm_supplFigure6.pdf108KSupplemental Figure 6. SMO silencing reduces melanoma cell growth. A) qRT-PCR analysis of SMO, GLI1 and PTCH1 in A375 cells transduced with LV-c, LV-shSMO-64 or LV-shSMO-65. qRTPCR values reflect ct values after normalization with 2 housekeeping genes (GAPDH and βACTIN) and are shown as fold change expression in each LV-shSMO vs LV-c. B-C) Growth curves of SSM2c (B) and A375 (C) cells transduced with LV-c, LV-shSMO-64 or LV-shSMO-65 lentivectors. Three to five thousands transduced cells/well were plated in 12-well plates and cells were counted on days 3, 5 and 7. Error bars represent SEM.
SC_12-0080_sm_supplFigure7.pdf105KSupplemental Figure 7. GLI1 silencing reduces melanoma cell growth. A) qRT-PCR analysis of GLI1 in A375 and SSM2c cells transduced with LV-c, LV-shGLI1-85 or LV-shGLI1-87. qRT-PCR values reflect ct values after normalization with 2 housekeeping genes (GAPDH and βACTIN) and are shown as fold change expression in each LV-shGLI1 vs LV-c. B-C) Growth curves of SSM2c (B) and A375 (C) cells transduced with LV-c, LV-shGLI1-85 or LV-shGLI1-87 lentivectors. Three to five thousands transduced cells/well were plated in 12-well plates and cells were counted on days 3, 5 and 7. Error bars represent SEM.
SC_12-0080_sm_supplFigure8.pdf107KSupplemental Figure 8. qRT-PCR analysis of HH pathway components in patient-derived melanoma cells (SSM2c, M14, M21 and M26c) and A375 cell line. qRT-PCR values reflect ct values after normalization with 2 housekeeping genes and are shown as fold change in spheres vs. monolayer.
SC_12-0080_sm_supplFigure9.pdf154KSupplemental Figure 9. Activation of the HH pathway by inhibition of PTCH1 increases the fraction of ALDHhigh population and self-renewal ability of A375 cells. A375 melanomaspheres transduced with LV-c or LV-shPTCH1, as previously described [41], were dissociated and plated in 12 well plates at 1 cell/μl dilutions. After one or two weeks spheres were counted. A, B) Flow cytometry analysis of adherent A375 cells transduced with LV-c (control) (A) or LV-shPTCH1 (B). C) Increase in the number of spheres in A375 cells transduced with LV-shPTCH1.
SC_12-0080_sm_supplTable1.pdf42KSupplementary Table 1
SC_12-0080_sm_supplTable2.pdf64KSupplementary Table 2

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