Fig. 4

Nestin levels are reduced in drug resistant melanoma cells and are associated with targeted therapy response in patients. a A375 SEN and RES cells were harvested and lysed to extract total proteins to perform Western blot analyses with the indicated antibodies. Tubulin levels have been estimated for the protein equal loading. b A375 SEN and RES cells have been stained with an antibody PE mouse anti-nestin for 45 min to perform flow cytometry analyses. Non-stained cells were used as negative control. Black arrow indicates the peaks relative to RES cells (left graph). Quantitative analyses were calculated as “fold change” ± standard deviation (SD) compared to SEN cells considered as 1. Data were analyzed using CytExpert version 2.2 software. c, d Migration and invasion assays were performed as described above using A375 RES cells transiently transfected with a plasmid encoding for nestin (NES) or the relative empty control (CTR) for 48 h. Results have been quantified by counting cells remaining on the top side of the membrane and the average number ± SD of cells are reported as fold change respect to control (SCR) considered as 100. e The online software GEPIA 2.0 (http://gepia2.cancer-pku.cn/#index) was interrogated to unveil nestin prognostic value based on Skin Cutaneous Melanoma (SKCM) data from The Cancer Genome Atlas (TCGA). Patients have been stratified according to the mutational status and the Kaplan Meier (KM) curves represent the data relative to the BRAF-mutant subtype (n = 149). f The mRNA levels relative to nestin (NES) have been extracted from bulk RNA-seq data deriving from 18 melanoma patients before starting MAPKi therapy (GSE65185) deposited in Gene Expression Omnibus (GEO) database. Data have been used to plot KM curves to assess the predictive value of NES for therapy response (Discovery cohort). g NES levels were evaluated by immunohistochemistry (IHC) in 14 melanoma patients’ biopsies collected before starting MAPKi therapy. Data have been used to plot KM curves to assess the predictive value of NES for therapy response (internal validation cohort). High and low levels were assessed by considering positive and negative z-scores, respectively. The hazard ratio, Cox models and the log-rank p values were evaluated to plot KM curves