Pch110

Briefly, 2 × 10⁶ KU812 cells were transfected via electroporation with 1 µg of the different AGAP2-luciferase reporter plasmids or pGL4.10, and 1 µg of pCH110 (Amersham). Then cells were transferred into 24 well plates and left for 24 hours. When treated, the cells were allowed to recover for 4 hours and then the treatment was added to the culture medium, for 20 hours. 24 hours post electroporation, the cells were lysed and the luciferase activity analysed with the Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System (ThermoFisher) in accordance with the manufacturer’s instructions. Reporter activity was measured with FLUOstar Optima device (BMG Biotech). The luciferase activity was normalised to the corresponding β-galactosidase values.
2 × 10⁴ DU145 cells were seeded into 24 well plates and transfected with 0.5 µg of the relevant AGAP2–luciferase plasmid and 0.5 µg of pCH110 using the CalPhos Mammalian Transfection kit (Clontech). After 12 hours, the growth medium was replaced and the cells were allowed to recover for 24 hours. Treatments were provided at the end of the recovery period for an additional 24 hours and the luciferase activity measured as above. If curcumin was used, the growth media was changed to clear DMEM and a 10 µM curcumin treatment was carried out for 1 hour. Then 1 µM ATRA was added for the indicated times.

Regions containing JUPe, MYBL2e, or CCNE1e were amplified from OE19 genomic DNA using primers containing 20 bp overlap regions with the multiple cloning site of the pGL3 Promoter vector (Promega, E1761) for luciferase assays, or between the InFusion arms of the hSTARR_ORI vector (Addgene, 99296) (Supplementary file 11). Final vectors were assembled using HiFi assembly (NEB, E5520S) according to the manufacturer’s instructions to create plasmids containing JUPe, MYBL2e, or CCNE1e enhancer regions in either hSTARR_ORI (pAS5008-pAS5010) or pGL3- vectors (pAS5011-pAS5013). All recombinant plasmids are available upon request. Enhancer vectors were transfected using the Amaxa Nucleofector II (Lonza) with Cell Line NucleofectorTM Kit V (Lonza, VCA-1003) and program T-020, according to the manufacturer’s instructions. For luciferase assays, 250 ng of enhancer vector was co-transfected alongside 50 ng of pCH110 (Amersham). For STARR-qPCR, 800 ng of vector was transfected. Enhancer activity was assessed using the Dual-Light Luciferase & β-Galactosidase Reporter System (Thermo Fisher Scientific, T1003) according to the manufacturer’s instructions, or by RT-qPCR.