Glomax 96 microplate luminometer with dual injectors

A 500 bp minimal promoter fragment immediately upstream of the Sca1 transcriptional start site was PCR amplified from genomic DNA using the primers Forward 5′-TAAACGCGCACACGTTTCTC-3′ and Reverse 5′-GGCCAGCATCTGACCTCTTT-3′, and cloned into the pGL4 luciferase. Human embryonic kidney HEK 293T cells maintained under standard culture conditions were plated on 6-well plates (3.5×10⁵ cells per well). 24 hours after plating, the HEK-293Tcells were transiently transfected using Lipofectamine LTX (Invitrogen) with 2.5 µg of the following Firefly luciferase reporter plasmids (pGL4-Sca-1 1 µg of Renilla luciferase plasmid (transfection control), and 2.5 µg of empty vector (pcDNA3) or plasmids expressing full length Sox2 (WT-Sox2), or one of the mutant Sox2 (ΔTAD and ΔHMG)[44] (link). After 24 hr, cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega). The lysate was assayed for luciferase and Renilla activity using the GloMax 96 Microplate Luminometer with Dual Injectors (Promega, Madison, WI), according to the Dual-Luciferase Reporter Assay system kit protocol (Promega). The luciferase activity was calculated relative to the TK Renila. All reporter assays were performed in triplicate, and the bars in the figures denote the standard error of the mean (SEM).

NIH3T3 (ATCC) cells were plated in 6-well plates and transfected with siRNA as described above at least 16 h post-plating. 396 ng of Gli1-responsive Firefly luciferase reporter plasmid, 4 ng of a control Renilla luciferase reporter plasmid under the control of a constitutively active TK promoter, and 1 μg of pcDNA3.1+ empty vector were transfected into cells at least 6 h post-siRNA transfection using lipofectamine LTX with Plus reagent (Thermo Fischer Scientific) according to the manufacturer’s protocol. 16 h after DNA transfection, cells were recovered in fresh media for 24 h. Stimulation with the indicated ligand was performed in low-serum OptiMEM media (Thermo Fischer Scientific) for 24–36 h. Luciferase assays were conducted using the Dual Luciferase Reporter Assay System (Promega) and measured on a GloMax 96 Microplate Luminometer with Dual Injectors (Promega).