MOESM1 of Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer

Additional file 1: Figure S1: Verification of effective transduction knockdown of SIM2 via sh-RNA. Infection of MCF7 cells with the SIM2-shRNA construct results in decreased SIM2 protein levels in comparison to a nonspecific scrambled shRNA construct. Figure S2: Verification of NFkB promoter luciferase and SIM2 promoter luciferase assays in the HEK293 cell line. A. Luciferase activity in HEK293T cells co-transfected with 5x kB binding sites upstream of the luciferase gene (5x NFkB-luc) and NFkB p65 and/or SIM2s. (Diagram of promoter construct is shown above for reference.) B. Luciferase activity in HEK293T cells co-transfected with 5x NFkB-luc and NFkB p65 and/or SIM2s with its repression domain deleted (SIM2sΔR). C. SIM2 promoter activity in HEK293T cells co-transfected with SIM2 promoter upstream of the luciferase gene and increasing amounts of NFκB p65 (50ng,100ng, and 150ng). D. SIM2 promoter activity in HEK293T cells after co-transfection of promoter with control vector (pcDNA3), NFκB p65, and/or IκB-SR. E. SIM2 promoter activity in HEK293T cells co-transfected with SIM2 promoter upstream of the luciferase gene and 150ng NFκB p65 compared with the SIM2 promoter activity in HEK293T cells co-transfected with NFκB double mutant SIM2 promoter upstream of the luciferase gene. ANOVA and Student’s t-test was performed to test significance. A, B, C all significant at p<0.05, *p<0.05. Figure S3: Verification of PTGS2 in various breast cancer cell lines. PTGS2 expression in MCF7, DCIS.COM, and SUM159 parental breast cancer cell lines. ANOVA and Student’s t-test was performed to test significance. A, B, C all significant at p<0.05. Figure S4: No correlation between Tumor size and SIM2s or COX-2 gene expression. A. %SIM2s positive nuclei compared to tumor size (cm2). B. %COX-2 (M+S) compared to tumor size (cm2). Prism7 was utilized for statistical significance analysis. Two-tailed t-test was performed to test significance. Figure S5: SIM2 expression in TCGA Breast primary site and metastasis. Oncomine analysis of SIM2 expression in TCGA Breast primary site (n=529) compared to metastasis (n=3). t-Test performed to test significance, p-value=0.006. The Oncomine™ Platform (Thermo Fisher, Ann Arbor, MI) was used for analysis and visualization. For further information, refer to the terms of use. Table S1: Antibodies used in study. Table S2: Primer sequences used in study.