Supplementary material: CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is associated with resistance to androgen deprivation therapy, and an increase in the population of neuroendocrine (NE) differentiated cells. It is hypothesized that NE differentiated cells secrete neuropeptides that support androgen-independent tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. The cytochrome b561 (CYB561) gene, which codes for a secretory vesicle transmembrane protein, is constitutively expressed in NE cells and highly expressed in CRPC. CYB561 is involved in the α-amidation-dependent activation of neuropeptides and contributes to regulating iron metabolism which is often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression and maintenance of the highly aggressive NE phenotype in CRPC. In our study, we found that CYB561 expression is upregulated in metastatic and NE pr..., Protein expression data was evaluated using a western blot. In silico analysis of gene expression data was performed by examining the Bittner Multi-cancer clinical microarray data set (GSE2109) from the International Genomics Consortium Project for Oncology (www.intgen.org) for neuropeptide receptor mRNA expression across different clinical tumor types. Gene expression analysis was performed by reverse transcription-quantitative PCR (RT-qPCR). Data for gene expression analysis (normalized to 18s rRNA, GAPDH, or β-Actin (ACTB) transcript levels) were log10 transformed before statistical analysis. The basal and androgen-regulated gene expression data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Results from the transdifferentiation experiment were analyzed using two-way ANOVA to determine the main effects of CYB561 knockdown and transdifferentiation, after which Student’s unpaired t-test was done to determine the effect of transdifferentiation within an shRNA group..., , # Supplementary material: CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer

## Data

All data were analyzed using GraphPad Prism version 8.0. The statistical analyses included in each file are taken from the summary of results shown by GraphPad.

### Figure1E, F.xlxs

Data are presented as tables with table headings denoting the mRNA measured and column titles denoting the cell lines where the mRNA expression was measured. In some cases, the term \"NORM\" precedes the gene name indicating that the data shown are already expression values normalized to the housekeeping gene. **Statistical analyses**: Ordinary one-way ANOVA and Tukey's multiple comparisons were used. The summary of the results is included in the data file. A P < 0.05 was set to indicate significance.

### Figure2A, B, C, D.xlxs

Data are presented as tables with table headings denoting the cell line used and mRNA measured and column titles denoting the hormone treatment done. **Statist...