*4.5. CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS)*

To determine optimal hPL concentration in growth medium, MUG Mel3 Ph and MUG Mel3 NPh cells were each seeded at 10<sup>4</sup> cells/100 µL medium in a clear 96-multiwell plate and incubated for 72 h at 5% CO<sup>2</sup> and 37 ◦C. Twenty µL of PMS/MTS solution (Promega) was added to each well. After 2 h of incubation at 5% CO<sup>2</sup> and 37 ◦C, OD values were measured at 490 nm and 720 nm for correction wavelength on a CLARIOstar photometer (BMG Labtech, Ortenberg, Germany). This experiment was performed only once in six replicates and was conducted to define optimal hPL concentration for culturing MUG Mel3 cells.

#### *4.6. Immunocytochemistry (ICC)*

Specific melanoma cell markers were selected for the characterization of isolated primary melanoma cells. Cells were seeded in glass chamber slides (7 <sup>×</sup> <sup>10</sup><sup>4</sup> per chamber) and grown in complete medium (10% FBS) to a confluency of 80%. Before fixation, cells were washed with 1× Hanks Balanced Salt Solution (HBSS, Thermo Fisher Scientific, Waltham, MA, USA) and dried for 1 h at RT. Cells were fixed by consecutive wash steps with 4% formaldehyde (Donauchem, Vienna, Austria), 1× PBS (Medicargo, Uppsala, Sweden), ice cold methanol (Merck, Darmstadt, Germany), and ice-cold acetone (Merck). All following procedure steps were performed at RT. All slides were rehydrated in 1× TBE pH 7.5 with

0.1% Tween (Sigma, St. Lois, MO, USA) buffer for 3 min, which was also used as washing buffer in-between steps. Endogenous peroxide activity was quenched by hydrogen peroxide block (Thermo Scientific, Rockford, IL, USA) for 10 min. Ultra V Block from Thermo Scientific was applied to reduce nonspecific background staining, by 5 min incubation. Selected monoclonal primary antibodies were diluted as shown in Table S2. Negative controls of the same isotype (Dako, Glostrup, Denmark) were applied and incubated for 1 h. After washing, primary enhancer was added for 30 min, followed by HRP Polymer (Thermo Scientific) and AEC chromogen (Thermo Scientific) incubation for 15 min in the dark, and for 5 min, respectively. Finally, cells were counterstained with hematoxylin and blued with running hot tap water each for 5 min. Slides were mounted with aquatex (Merck) and analysed by an Olympus BX53 reflected-light microscope (Hamburg, Germany), 10× magnification, numerical aperture: 0.30 with an Olympus U-TV1X-2 T7 camera (Tokyo, Japan) at RT. Pictures taken were evaluated by the CellSens Standard 2 software (Olympus, Hamburg, Germany).

#### *4.7. qPCR*

Melanoma cells (2.2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells) were seeded onto six-well plates and incubated for 24 h. Subsequently, RNA isolation was performed with the Monarch® total RNA Miniprep Kit (New England Biolabs, Ipswich., MA, USA) according to the product manual. RNA quality and quantity were measured with a NanoDrop 2000 (Thermo Fisher Scientific). To remove melanin, RNA samples were further filtered using the OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine, CA, USA). cDNA was obtained from mRNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's manual. qPCR runs were performed on the CFX384 (Bio-Rad Laboratories Inc, Hercules, CA, USA) using primers against Melan-A and Sry-related HMG-Box gene 10 (SOX10; Table S3), and the 5X HOT FIREPol EvaGreen qPCR Supermix (Solis BioDyne, Tartu, Estonia). Results were analysed using 2−(∆∆Ct) values. Means of GAPDH and β-Actin (ACTB) as reference genes were used for calculation of ∆Ct values. Three independent biological experiments were conducted each in triplicates.

#### *4.8. FACS Analysis*

<sup>1</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> cells each were harvested from 80–90% confluent cultures and stained in 100 µL FACS buffer with 2.5 µL V450 mouse anti-Human CD271 antibody Clone C40-1457 (BD Horizon, Heidelberg, Germany) for 30 min at 4 ◦C. Cells were measured on a CytoFLEX S Flow Cytometer (Beckman Coulter, IN, USA) and further analysed using the Software CytExpert 2.3 (Beckman Coulter). The percentage of CD271 positive cells was calculated based on unstained controls and FSC/SSC gating was used to exclude debris, dead cells and doublets. The A375 cell line served as a positive control and Hela cells as negative controls (data not shown). Experiments were run in three independent experiments.
