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. Author manuscript; available in PMC: 2016 Dec 1.

Published in final edited form as: Mol Cancer Res. 2015 Sep 15;13(12):1602–1614. doi: 10.1158/1541-7786.MCR-15-0213

TSC2 inhibits NF-κB transcription and target gene expression in PC3 cells. A. PC3 cells were transfected with control siRNA or siRNA to TSC2 for 24 hours then followed by transfection with empty vector plus 200 ng of 3× κB luciferase reporter and 30 ng of pRL-SV40 (Renilla reporter control). Cells were harvested after 24 h and luciferase assays were performed. Three independent experiments were performed, each in triplicates. B. PC3 cells were transfected with siRNA control, RheB or Raptor for 24 hours and then transfected with the NF-κB luciferase reporter for another 24 hours and Luciferase assays were performed as in (A). Three independent experiments were performed, each in triplicates. C. PC3 cells were transfected with siRNA control, TSC2, TSC2 plus RheB, or TSC2 plus Raptor as indicated for 24 hours then followed by transfection of 200 ng of 3×κB luciferase reporter and 30 ng of Renilla reporter control. Cells were harvested and luciferase assays were performed. D. PC3 cells were transfected with siRNA control or TSC2 as indicated. RNA was extracted 48 h after transfection and RT–PCR was performed to assess changes in mRNA levels of NF-κB target gene expression. E. PC3 cells were transfected with siRNA control or TSC2 as indicated. Cells were treated with DMSO or IKKβ inhibitor (5μM) for another 48 hours and cell proliferation was measured by MTT assay. (* P<0.05, ** P<0.01, *** P<0.001).

TSC2 inhibits NF-κB transcription and target gene expression in PC3 cells. A. PC3 cells were transfected with control siRNA or siRNA to TSC2 for 24 hours then followed by transfection with empty vector plus 200 ng of 3× κB luciferase reporter and 30 ng of pRL-SV40 (Renilla reporter control). Cells were harvested after 24 h and luciferase assays were performed. Three independent experiments were performed, each in triplicates. B. PC3 cells were transfected with siRNA control, RheB or Raptor for 24 hours and then transfected with the NF-κB luciferase reporter for another 24 hours and Luciferase assays were performed as in (A). Three independent experiments were performed, each in triplicates. C. PC3 cells were transfected with siRNA control, TSC2, TSC2 plus RheB, or TSC2 plus Raptor as indicated for 24 hours then followed by transfection of 200 ng of 3×κB luciferase reporter and 30 ng of Renilla reporter control. Cells were harvested and luciferase assays were performed. D. PC3 cells were transfected with siRNA control or TSC2 as indicated. RNA was extracted 48 h after transfection and RT–PCR was performed to assess changes in mRNA levels of NF-κB target gene expression. E. PC3 cells were transfected with siRNA control or TSC2 as indicated. Cells were treated with DMSO or IKKβ inhibitor (5μM) for another 48 hours and cell proliferation was measured by MTT assay. (* P<0.05, ** P<0.01, *** P<0.001).