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 3 experiments. Table S1. List of primers used in the studies. Table S2. Prostate cancer finder RT2 Profiler PCR array data. (ZIP 2150 kb)

补充图S1：前列腺癌（Prostate Cancer, PCa）中生长激素促分泌素受体的表达
A. 高危前列腺癌患者活检样本（n=20）及接受膀胱前列腺切除术患者的正常前列腺组织样本（n=7）中生长激素促分泌素受体1a（GHSR1a）与生长激素促分泌素受体1b（GHSR1b）的mRNA表达水平。表达水平通过实时荧光定量聚合酶链反应（quantitative real-time PCR, qPCR）测定，并以由肌动蛋白β（ACTB）和甘油醛-3-磷酸脱氢酶（GAPDH）表达计算得到的标准化因子（NF）进行校正。数据采用曼-惠特尼U检验进行分析，结果以中位数（四分位距，IQR）表示；
B. 前列腺癌细胞系中GHSR1a与GHSR1b的mRNA表达：其中雄激素依赖性细胞系包括VCaP、22Rv1，去势抵抗性细胞系包括PC-3、DU145。不同传代细胞（n≥3）的mRNA绝对水平通过qPCR测定，并以ACTB进行标准化；数据以DU145（GHSR1a）和VCaP（GHSR1b）的表达为100%的百分比形式呈现，因二者为对应受体表达最低的细胞系，此举旨在便于不同细胞系间的比较；
C. 使用多对引物对不同前列腺癌细胞系（22Rv1、DU145、LNCaP、PC-3）、正常类前列腺细胞系（EPN）及前列腺癌样本中的GHSR1a表达进行进一步分析，以表达GHSR1a的垂体瘤cDNA作为阳性对照。

补充图S2：LNCaP细胞经生长激素释放肽基因衍生肽处理后睾酮分泌及雄激素受体（Androgen Receptor, AR）系统的调控
A. 用天然生长激素释放肽（native-ghrelin）或In1-ghrelin肽（10 nM）处理24小时后，培养基中的睾酮水平；
B. 用生长激素释放肽或In1-ghrelin衍生肽（10 nM）处理24小时后，AR的mRNA表达水平；
C. 用天然生长激素释放肽或In1-ghrelin肽（10~30 min）处理后，磷酸化AR（Ser81）的时间依赖性激活情况：磷酸化AR（Ser81）的蛋白水平以总AR进行标准化，展示了LNCaP细胞系的代表性免疫印迹结果；
D. 用天然生长激素释放肽或In1-ghrelin肽（10 nM）处理24小时后，前列腺特异性抗原（Prostate Specific Antigen, PSA）的分泌及mRNA表达水平。mRNA绝对水平通过qPCR测定并以ACTB标准化，数值以均值±标准误（standard error of the mean, SEM）表示，实验重复次数n>3。

补充图S3：通过qPCR验证PC3细胞中In1-ghrelin与生长激素释放肽过表达的代表性结果
mRNA绝对水平通过qPCR测定并以ACTB标准化，数值以均值±标准误表示，实验重复次数n>3。星号代表显著性差异。

补充表S1：本研究所用引物列表
补充表S2：前列腺癌Finder RT2 Profiler PCR芯片阵列数据（ZIP格式，大小2150 kb）