Cells were washed with PBS, incubated with appropriate secondary antibodies, nuclear stain Hoechst, filamentous actin stains Texas Red Phalloidin promotion information or 488 Phalloidin and Cell Mask Blue for 4 hrs at room temperature. Cells were Inhibitors,Modulators,Libraries washed then imaged using PerkinElmer Opera Confocal Imager and an Olympus IX 81 Scanning Confocal microscope. Proliferation assays of mono and co cultured 3D cells To assess cell proliferation in mono and co cultured 3D cells, assays were performed in 384 well plates using Alamar Blue reagent. TC treated Falcon 384 well plates were applied with 15 ul of 70% Matrigel and left to polymerise for 2 hrs at 37 C, 5% C02 and 95% humidity. Mono culture were plated at 800 cellswell and co cultures were plated at 400 cellswell each to make a total of 800 cellswell in 50 uL complete medium per well and left to adhere ON at 37 C, 5% CO2 and 95% humidity.

A baseline reading was taken 24 hours after plating, and readings were obtained on assay days 3, 6 and 9 through application of 5 ul Alamar Blue per well, Inhibitors,Modulators,Libraries achieving a final concentration of 10%. Inhibitors,Modulators,Libraries After addition of Alamar Blue cells were further incubated for 4 hrs at 37 C, 5% CO2 and 95% humidity, before plates were read on the Envision Plate Reader using fluorescence excitationemission Inhibitors,Modulators,Libraries settings of 530 nm 595 nm. To investigate Inhibitors,Modulators,Libraries the relative contribution of prolifer ating HS5 and PC3 cells in co culture, cells were treated with Click iT EdU HCS 594 kit at days 3, 6 and 9 in culture. After incubation with the EdU compound in serum free media, cells were fixed with PFA, washed and a 594 fluorescent azide solution was applied ON at 4 C in blocking buffer along with STRO 1 antibody.

The following day a general cytoplasmic Compound C and nuclear stain and a secondary antibody was ap plied for 4 hrs at RT. Cells were finally washed and imaged using an Olympus confocal and results were analysed using Imaris volume and spots. Transwell cell invasion assays To investigate the role integrin 6 and B1 play in medi ating invasive cell behaviour, transwell cell invasion as says were employed. Two days prior to each invasion assay, PC3 and HS5 cells were seeded in 6 well plates at a density of 500,000 cellswell and co culture cells were seeded together at a 1 1 ratio to a total 500, 000 cellswell and left to adhere ON at 37 C, 5% C02 and 95% humidity. The following day, cultures were serum starved for 16 24 hours in the presence of integrin function blocking antibodies 1. 5 ugmL of 6 GoH3, 1. 5 ugmL of B1 P5B2, 6 and B1 and 1. 5 ugmL of mouse IgG isotope controls. On the day of the assay, cells were harvested with accutase and seeded at a density of 150,000 cells per transwell insert in a volume of 200 ul SFM with the addition of integin inhibitors 1. 1.