Lightswitch luciferase reagent

ChIP was performed on confluent Caco2 IECs as previously described (Clambey et al., 2012 (link); Glover et al., 2013 (link)). Briefly, cells were exposed to either normoxia or hypoxia for 6 h and fixed in 1% formaldehyde for 10 min at 4°C. Cells were sonicated to shear genomic DNA and incubated overnight with 5 μg of either control Rb IgG or Rb pAb against HIF1α (NB100-134; Novus Biologicals, Littleton, CO). The resulting complexes were precipitated using Fastflow G-Sepharose beads (GE Healthcare, Little Chalfont, United Kingdom), eluted, purified, and analyzed by PCR using HRE spanning primers (Supplemental Table S1).
CLDN1 promoter luciferase reporter plasmid (Switchgear Genomics, Carlsbad, CA) was mutated using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) with primers against both HRE sites. Using Lipofectamine LTX (Invitrogen), 100 ng of plasmid was transfected into subconfluent HeLa cells and luciferase activity determined at 24 h using LightSwitch luciferase reagent (Switchgear Genomics) and luminescence determined using the Glomax Multi plate reader (Promega).

LNCaP cells were seeded in triplicate in a 96-well plate and co-transfected the following day with a luciferase reporter plasmid containing either wild-type MIB1 3′UTR or MIB1 3′UTR containing mutations to disrupt binding in the miR-198 predicted binding site (SwitchGear Genomics; Active Motif), and control or miR-198 mimic. After 24 h, luciferase activity was assayed according to the manufacturer's protocol using LightSwitch Luciferase Reagent (SwitchGear Genomics; Active Motif).