Additional file 1: of The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness

Figure S1. Ghrelin receptor expression in PCa. A. GHSR1a or GHSR1b mRNA expression in biopsies from patients with high-risk PCa (n = 20) and normal prostate samples from patients that underwent cystoprostatectomies (n = 7). Expression levels were determined by qPCR and adjusted by a normalization factor (NF) calculated from ACTB and GAPDH expression levels. Data were evaluated by Mann-Whitney test and represent median (IQR); B. GHSR1a or GHSR1b mRNA expression in PCa cell lines (androgen-dependent: VCaP, 22Rv1 and castration resistant: PC-3, DU145). Absolute mRNA levels from different passages (n ≥ 3) were determined by qPCR and adjusted by ACTB [Data are expressed as percent of DU145 (GHSR1a) and VCaP (GHSR1b) set at 100%, since they are the cell lines with lesser expression of those receptors, and, in order to ease the comparison between the cell lines); C. Further analysis of GHSR1a expression on different PCa cell lines (22Rv1, DU145, LNCaP, PC-3), normal-like prostate cell line (EPN) and PCa samples using several pairs of primers. As positive control, cDNA from a pituitary tumor with GHSR1a expression was used. Figure S2. Secretion of testosterone and modulation of androgen receptor (AR) system after treatment with ghrelin gene-derived peptides in LNCaP cells. A. Testosterone levels in the culture medium after 24 h of treatment with native-ghrelin or In1-ghrelin peptides (10 nM); B. AR mRNA expression after 24 h of treatment with ghrelin or In1-ghrelin derived peptides (10 nM); C. phospho-AR (Ser81) time-course activation after treatment with native-ghrelin or In1-ghrelin peptides (10–30 min). Protein levels of phospho-AR (Ser81) were adjusted by total AR. Representative blots in LNCaP cell line are showed; D. PSA secretion and mRNA expression after 24 h of treatment with native-ghrelin or In1-ghrelin peptides (10 nM). Absolute mRNA levels were determined by qPCR and adjusted by ACTB. Values represent mean ± SEM of n > 3 experiments. Figure S3. Representative figures showing the validation of In1-ghrelin and ghrelin overexpression in PC3 cells by qPCR. Absolute mRNA levels were determined by qPCR and adjusted by ACTB. Values represent mean ± SEM of n > 3 experiments. Asterisk indicate significant difference (***p < 0.001). Figure S4. Grade of inflammation in xenografted PC3-derived tumors. Representative images of hematoxylin–eosin (H/E) staining are depicted. Tumors derived from native ghrelin stably-transfected PC-3 cells presents more inflammation (observed as darker foci in the pictures) compared to In1-ghrelin or mock derived tumors. Asterisk indicate significant difference (*p < 0.05). Figure S5. Expression of native-ghrelin in LNCaP and PC-3 cells in response to In1-ghrelin silencing by specific siRNA. Absolute mRNA levels were determined by qPCR and adjusted by ACTB. Values represent mean ± SEM of n > 3 experiments. Table S1. List of primers used in the studies. Table S2. Prostate cancer finder RT2 Profiler PCR array data. (ZIP 2150 kb