Gene-specific regulation of NF-κB target genes by CDK6.

<p>(<b>A</b>) HEK293IL-1R cells were transiently transfected with a vector directing the synthesis of two different shRNA duplexes directed at CDK6 or an empty vector control along with a NF-κB-dependent reporter gene and the SV40-ß gal vector to allow for normalization. After 24 h, cells were stimulated for 4 h with IL-1α(10 ng/ml, black bars) or left untreated (white bars). Cells were lysed and luciferase activity was normalized for co-transfected SV40-promoter driven ß-galactosidase. The graph shows the mean luciferase activity +/− s.e.m. from three independent experiments performed in duplicates relative to the vector control. (<b>B</b>) HEK293IL-1R cells were transiently transfected with empty vector, NF-κB (3) luc and SV40-ß gal. After 24 h cells were pretretreated with 10 µM PD332991 and then further stimulated for 4 h with IL-1α(10 ng/ml) or left untreated. Shown is the mean luciferase activity +/− s.e.m. from two independent experiments. (<b>C</b>) HeLa Fucci cells were arrested for two days and then left untreated or were treated with 10 µM PD332991 (hatched bars) for 30 min followed by 30 min TNFα (20 ng/ml) as indicated. Thereafter, total RNA was prepared and analysed for the expression of the indicated mRNAs by RT-qPCR. Data show the mean –fold change +/− s.e.m. from two independent experiments performed in duplicate. The asterisk indicates significant differences (p< = 0.05) as determined by paired t-test.