Fig. 1

Screening for small molecules that interfere with Bcl-3-mediated cyclin D1 activity. A Survival curve of melanoma patients with metastasis stratified by Bcl-3 expression level. Patients are categorized into two groups: Bcl-3 high (positive z scores) and Bcl-3 low (negative z scores), based on the calculated z-scores for Bcl-3 expression from three pivotal studies. The x-axis represents the survival time in months, while the y-axis depicts the proportion of patients surviving. The survival differences between the two groups were evaluated, providing insights into the impact of Bcl-3 expression levels on the survival of melanoma patients with metastases. B Lysates prepared from human melanoma cell lines were subjected to Western blot analysis of Bcl-3 and α-Tubulin. C The effects of Bcl-3 on cyclin D1 promoter activity. Mel Juso cells were cotransfected with empty vector (EV), Bcl-3, cyclin D1 κB mutant, together with reporter plasmid cyclin D1 luciferase and Renilla-luciferase. Cyclin D1 promoter activity was measured using the Dual Reporter Luciferase assay. D Schematic overview of the cell-based luciferase reporter assay as a high throughput screening model. This model utilizes a reporter construct containing only the part of the cyclin D1 promoter (-64/ + 10) that can be occupied by Bcl-3, or a cyclin D1 κB mutant as a control. The upper panel shows the Bcl-3-specific κB site on the cyclin D1 promoter. The lower panel shows the ligands that were selected that could inhibit Bcl-3-mediated cyclin D1 promoter activation. E Small molecule screening using 1368 small molecules from a chemical library from the National Cancer Institute. Mel Juso cells were transfected for 24 h with Bcl-3 and cyclin D1, followed by treatment with 10 μM of each small molecule. After 24 h of treatment, samples were analyzed using a DLR luciferase assay. Ligands were selected based on the selection criterion of a 50% or greater reduction in Bcl-3-mediated cyclin D1 promoter activity. F Mel Juso cells were transfected with Bcl-3 and cyclin D1 κB mutant constructs and subjected to treatment with the 79 selected ligands from the first screen. The selection criterion for screen 2 was that no reduction in cyclin D1 promoter activity could be observed in the presence of the cyclin D1 κB mutant. G Mel Juso cells were transfected with Bcl-3 and cyclin D1 (-64/ + 10) constructs and treated with 15 ligands (selected after screen 1 and screen 2). Bcl-3-mediated promoter activity in the presence of each ligand was analyzed using the DLR luciferase assay. All data are presented as the mean ± SEM from three independent experiments. Statistical significance was assessed using Student’s t test