Fig. 3

Characterization of the Bcl-3 antagonist that inhibits cyclin D1 expression. A Mel Ju cells were transfected with cyclin D1 or cyclin D1 κB mutant promoter together with Renilla Luciferase. Following transfection, cells were treated with 50 μM BCL3ANT for 24 h and analyzed for Bcl-3-mediated cyclin D1 promoter activity using the luciferase assay. B Mel Juso cells were cotransfected for 24 h with the Bcl-3 and cyclin D1 promoters, followed by 2a 4-h treatment with 10 μM, 20 μM, or 30 μM BCL3ANT, or DMSO as a control. Following incubation with treatment, Bcl-3-mediated cyclin D1 promoter activity was analyzed by luciferase assay. C The half-maximal inhibitory concentration (IC₅₀) of Mel JU was determined after measuring cell viability using a WST-1 assay. D (Left) mRNA from Mel Ju cells was prepared after 24 h of treatment with or without 50 μM BCL3ANT and subjected to real-time quantitative PCR (qPCR) analysis of cyclin D1 and the housekeeping gene GAPDH. (Right) Lysates prepared from Mel Ju cells following 24-h treatment with or without 50 μM BCL3ANT were subjected to Western blot analysis of cyclin D1 and α-tubulin. E mRNA from Mel Ju cells was prepared after 24 h of treatment with or without 50 μM BCL3ANT and subjected to real-time quantitative PCR (qPCR) analysis of Bcl-3 and the housekeeping gene GAPDH. F SiRNA-induced knockdown of Bcl-3 or siRNA-control knockdown cells in immortalized keratinocytes were treated with DMSO or 50 μM BCL3ANT for 24-96 h before being subjected to a WST-1 assay to measure cell proliferation. Data are presented as the mean ± SEM from three independent experiments. Statistical significance was assessed using Student’s t test