Mithras lb941

The luciferase reporter vectors, the OCT4, ICAM3 gene promoter fragments were amplified from human genomic DNA and cloned into the firefly luciferase plasmid pGL3-basic-IRES. For reporter assays, above constructs along with pRL-TK plasmid (internal reference) were co-transfected into MDA-MB-231 or A549 cells. Then cells lysates were collected and analyzed with Dual-luciferase reporter assay system following its manual (Promega, E1910). And the reporter activity was measured using a microplate reader (Mithras LB941, Berthold Technologies).

The human siGENOME SMART pool library targeting 1027 inflammation-related genes were obtained from Dharmacon. For the siRNA screens, transfection mixtures were 50nM siRNA, 100ng OCT4 reporter constructs (QIAGEN) and 0.3 ul Lipofectamine 2000 (Invitrogen) in 50 ul opti-MEM medium (Invitrogen), three replicates were used for each siRNA. In each plate, we used both POU5FI (5460) siRNA-SMART pool Dharmacon M-019591-03-0005 and SiGENOME Human PRDM14 (63978) siRNA-SMART pool as positive controls [21 (link)]. HMLE-snail cell were plated in 50ul opti-MEM medium with a density of 10000 cells per well in 96-well plate. Dual Luciferase reporter assay was carried out for medium-throughput readout (Promega), and the level of OCT4 reporter gene expression was measured using a microplate reader (Mithras LB941, Berthold Technologies). OCT4 reporter values were normalized to the expression of the Renilla luciferase reporter. Fold change value of each siRNA sample was calculated. The distribution of the datasets was determined to be closed to normal distribution, so the Student pair-t test between each siRNA group and the relative negative control group was performed. The cut-off values for the screen are Fold change <0.5 or >2.0, and P<0.05.