Dual light luciferase β galactosidase reporter gene assay system

HepG2 cells were co-transfected with the plasmids of HNF4α-specific luciferase reporter ApoB-luc and β-Galactosidase. 24 h upon transfection, the cells were treated with EMEM containing 10% of sera collected from the tail veins of the WT or FKO mice. Upon the serum treatment for 18 h, the cells were harvested for the luciferase assay. Briefly, the luciferase activities in the lysed cells were determined by Dual-Light Luciferase & β-Galactosidase Reporter Gene Assay System (Thermo Fisher Scientific), and normalized to β-galactosidase activity.

The cells were rinsed three times with 1× PBS and lysed with 130µL TROPIX lysis solution (Thermo Fisher Scientific) added 0.5 mm DTT by incubation for 10 min on ice. Ten microlitre of lysate was transferred to a 96‐well light plate, whereafter luciferase activity and β‐galactosidase activity were measured using 5‐s integration time and 2‐s delay using the Dual‐Light™ Luciferase & β‐Galactosidase Reporter Gene Assay System (Thermo Fisher Scientific) in GloMax luminometer.

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.

B35 neuroblastoma cells were plated in a 96-well plate (Celltreat Scientific Products) at a concentration of 2.0×10⁵ cells/ml and allowed to adhere overnight. The next day, cells were co-transfected with the appropriate Luciferase 3′ UTR reporter plasmid, β-Galactosidase control, and 100 nM of miR-200a, miR-200b or control mimic (Qiagen) per well using Lipofectamine 3000 (Invitrogen). After 48 h, luciferase activity was determined using Dual Light Luciferase & β-Galactosidase Reporter Gene Assay System (ThermoFisher Scientific) according to the manufacturer's protocol.

Wild-type (wt) and mutant (mut) seed regions of the target genes (PTEN, TXNIP, TSC1 and CAV1) were constructed and cloned into Luc 3′UTR between the HindIII and BcuI sites of pMIR-REPORT-Luciferase vector (Ambion by Thermo Fisher Scientific, Grand Island, NY, USA). The oligonucleotide sequences are listed in Supplementary Table S2. Constructed vectors were verified by Sanger sequencing using Applied Biosystems^® 3500 analyzer (Applied Biosystems, Foster City, CA, USA). AGS cells (100,000 cells/well) were co-transfected with 146 ng of constructs (wt or mut vector) and with 50 nM of either miRNA mimic or negative mimic control using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). After 48 h incubation luciferase activity was detected by Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System (Applied Biosystems, Foster City, CA, USA) following manufacturer’s protocol. Luminescent signal was quantified by GENios Pro microplate reader (Tecan Trading AG, Mannedorf, Switzerland). Reporter activity was normalized to β-Galactosidase activity.