Lb960 luminometer

Cells were transiently transfected with 2 μg of firefly/renilla luciferase reporter plasmid. Protein extracts were prepared using the Passive Lysis Buffer provided in the Dual-Luciferase Assay (Promega). Equal amounts of protein extracts were plated into a 96-well plate. Firefly luciferase activity was measured for 12 seconds using the LB 960 luminometer (Berthold Technologies, Thoiry, France). To assess the internal standard activity, Stop and Glo reagent was added (Promega), and the peak of the renilla luciferase activity was then measured. Normalized relative luciferase units (RLU) were then calculated as firefly luciferase units of protein extracts of treated or untreated cells divided by renilla luciferase units of protein extracts of untreated cells. Data represent the mean ± SEM of three independent experiments, each performed in duplicate.

Briefly, HEK-293 cells were seeded into 12-well plates at a density of 5 × 10⁴ per well the day before transfection. Transfection master mixes were prepared for triplicate wells each receiving 500 ng pGL-Foxp3-P^{25 (link)} and a 375 ng sum of balancing pSG5 empty vector with 125, 250, or 375 ng of pSG5-SRC-3 plasmid expressing the human SRC-3 coding sequence. Approximately 24 h after transfection, cells were harvested in 1X Passive Lysis Buffer (Promega) and quantified using Promega Luciferase Assay System (cat. E1500). Measurements were read on a Berthold LB 960 luminometer.

HUVECs (2×10⁴) were seeded in 24-well plates and cultured overnight. NRP1-promoter (0.5 μg) was transfected using Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h of transfection, AS (1 μM) and/or IL-13 (50 ng/mL) were added to the media, and the 24-well plate was incubated at 37°C in a humidified atmosphere for an additional 24 h. HUVECs in the 24-well plates were lysed for luciferase assay. Luciferase and Renilla activities were determined by a Luciferase-Renilla assay system (E1980, Promega) on an LB960 luminometer (Berthold, Germany).

To construct the EPO enhancer-driven luciferase reporter gene, the human EPO enhancer region (5-GGTACCGGCCCTACGTGCTGTCTCACACAGCCT GTCTGACCTCTCGACCTACCGGCCAGATCT-3) was inserted into the multi-cloning site of the pGL4 vector (Promega, Madison, WI). Mutated EPO luciferase was generated by altering the DNA sequence targeted by HIF-1 (CGTG to AAAA). Hep3B cells were transfected with 1 mg of the reporter plasmid using Lipofectamin 2000 (Life Technologies). After stabilized for 48 hours, the Hep3B cell lines stably harboring the reporter plasmid were selected under G418, and five stable cell lines were mixed to rule out the artifact generated by the integration of the plasmid into chromosomes. The cell line was pre-treated with each compound in a chemical library for 4 hours, and then incubated under normoxia or hypoxia for 16 hours. Cells were lysed and luciferase activity was measured using a LB960 luminometer (Berthold Technologies, Oak Ridge, TN). To evaluate the cap-dependent and the IRES-dependent translation of HIF-1α mRNA, we used thymidine kinase (TK) promoter/HIF-1α 5′-UTR/luciferase and CMV promoter/GFP/HIF-1α 5′-UTR/luciferase reporters, respectively [41 (link)]. The β-gal expression plasmid was cotransfected into cells to normalize transfection efficiency.