Fig 2: (Related to Fig 5) Effect of BACE1/2 inhibition and BACE2 KD in IGR37 metastatic melanoma cells and of PMEL administration in IGR39 primitive cells AConfocal fluorescence images of anti-YAP antibody (green) and DAPI staining (blue) in IGR37 upon treatment with DMSO or BACE1/2 inhibitor. Scale bar is 10 µm.BBACE1 and BACE2 mRNA levels measured by real-time PCR in IGR37 and IGR39. N = 3 biological replicates. T-test, *P < 0.05, **P < 0.001. Data are mean ± SD.CBACE2 mRNA levels measured by real-time PCR in iBACE2 KD IGR37 treated or not with doxycycline. N = 4 biological replicates. T-test analysis, **P < 0.001. Data are mean ± SD.DWestern blot and densitometric analysis of BACE2 and PMEL Ma relative expression in iBACE2 KD IGR37 treated or not with doxycycline. Actin was used as loading control. N = 4 biological replicates. T-test, **P < 0.01, ****P < 0.0001. Data are mean ± SD.EVolcano plot of the proteins secreted by iBACE2 KD IGR37 cells treated or not with doxycycline (Secret3D workflow). N = 4 biological replicates. Proteins downregulated upon BACE2 silencing are reported in the right part of the plot. Venn diagram of statistically significant proteins common between NB-360 treatment and BACE KD in IGR37 (right upper corner).FConfocal fluorescence images of Proteostat signal (1:2,000, red) and DAPI staining (blue), scale bar is 10 µm, and quantitation of protein aggregates in iBACE2 KD IGR37 cell lines treated or not with doxycycline by immunofluorescence analysis using Fiji software. N = 8 biological replicates. T-test, ****P < 0.0001. Data are mean ± SD.GmRNA levels of CTGF measured by real-time PCR in iBACE2 KD IGR37 treated or not with doxycycline. N = 3 biological replicates. T-test, *P < 0.05. Data are mean ± SD.HConfocal fluorescence images of Proteostat signal (1:2,000, red) and DAPI staining (blue), scale bar is 10 µm, and quantitation of protein aggregates in IGR cell lines treated or not with 3I inhibitor by immunofluorescence analysis using Fiji software. N = 14 biological replicates. T-test, ****P < 0.0001. Data are mean ± SD.ImRNA levels of CTGF measured by real-time PCR in IGR37 treated or not with 3I inhibitor. N = 3 biological replicates. T-test analysis, *P < 0.05. Data are mean ± SD.JmRNA levels of Agrin measured by real-time PCR in IGR39 treated with recombinant PMEL. N = 3 biological replicates. T-test, **P < 0.01. Data are mean ± SD.KWestern blot and densitometric analysis of pFAK(Y397) in IGR39 treated with recombinant PMEL. Actin was used as loading control. N = 3 biological replicates. T-test, **P < 0.01. Data are mean ± SD. Source data are available online for this figure.

Fig 3: Oxysterol quantification in primitive and metastatic melanoma cells and PMEL expression in IGR37 and IGR39 melanoma cell line AAbsolute quantitation of 24-, 25- and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***P < 0.001, N = 3 biological replicates, data are mean ± SD).BHistogram representing R square of correlation analysis between absolute quantitation of 24-, 25- and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines.CConfocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10 µm.DWestern blot on IGR37 and 39 cellular lysates probed with anti-HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates. Source data are available online for this figure.

Fig 4: Gradual loss of melanoma cells along culture passages. (A) Melan-A expression of the patient-derived cells from the first passage revealed high purity of melanoma cells. (B) Alpha-smooth muscle actin (a-SMA) and Melan-A expression of the patient-derived cell culture from the third passage. Melanoma cells tended to grow on the layer of fibroblasts. (C) Expansion of fibroblasts in the older passage—expression of fibroblast activation protein (FAP) and HMB45, a melanoma marker, of the patient-derived cell culture from the ninth passage.