BGISEQ-500 paired end sequencing; The cross-talk between prostate cancer cells and myeloid cells to prime cancer metastasis
The cross-talk between prostate cancer cells and myeloid cells to prime cancer metastasis
PCa cells were stained with CFSE (Invitrogen, Sweden), or DiD (Molecular Probes Inc. OR, USA) for 15 min according to manufacturers' protocols prior to co-culture. Sub-populations of PC-3 cells and U-937 cells that were double positive or single positive to DiD and CFSE were sorted and isolated by using FACSAria III (BD Biosciences, USA). The sorted subpopulations were confirmed by using fluorescent microscopy. Flow cytometry analysis was performed using CytoFLEX and CyAn™ ADP (Beckman Coulter, USA) analysers. Data was analyzed using FCS Express (DeNovo Software, USA), FlowJo (Tree Star, Inc., USA) and CytExpert (Beckman Coulter, USA) softwares.  PC-3 and U-937 cells were purchased from American Type Culture Collection (Manassas, VA, USA). An androgen-insensitive cell line, PC-3M (29), was kindly provided by Dr. J Fidler (Department of Urology, MD Andersson Cancer Center, TX, USA). PC-3, U-937 and bone marrow cells in mono- or co-culture were treated with ISA-2011B at 25 µM or solvent DMSO (0.125%).PC-3/PC-3M cells and U-937 cells or bone marrow cells were cultured in mono- or co-culture at different ratios for 1, 6, 24 and up to 48 hours. For co-culturing of PCa cells in conditional media, the media contains 50% supernatant of U-937 cells in RPMI-1640 with 10% FBS. For proliferation assay, PCa cells were stained with CFSE (Invitrogen, Stockholm, Sweden) before subjecting to co-culture or mono-culture at various time point according to manufacturer's instruction. BGI standard method A total of 1 μg of RNA/ sample were used for library preparation