File S1 - Reduced Selenium-Binding Protein 1 in Breast Cancer Correlates with Poor Survival and Resistance to the Anti-Proliferative Effects of Selenium

Supporting figures. Figure S1 Reduced SELENBP1 expression in hyperplastic enlarged lobular analyzed from public microarray database. The raw data from the microarray study were downloaded from NCBI Gene Expression Omnibus (GEO) or EBI ARRAYEXPRESS. The gene information and annotation of data sets were downloaded from the manufacturer of the microarray. The raw data and array information were inputted into dchip analysis software. After normalization and modeling, expression values (mean fluorescent intensity) were exported. Statistical analysis and boxplot graph were performed in SPSS software. *denotes p+ MCF7 cells were transfected with either scrambled control or ERα-specific siRNA duplexes by Lipofectamine 2000. ER expression levels were determined by western blot at 48 h after transfection. Figure S4 Downregulation of SELENBP1 expression in MCF-7 cells upon estrogen treatments analyzed from public microarray database. Microarray data analysis was performed as described previously. (A) A time-dependent reduction of SELENBP1 expression upon E2 treatment. Dunnett T3 test was performed to show p values compared with each time point. The data sets are from GSE11324. (B). Reduction of SELENBP1 expression up to 48 h of E2 treatment. The data sets are from GSE11352. Figure S5 Determination of ER expression after ER plasmid transfection. ER– SKBR3 and MDAMB453 cell lines were transfected with ER expression plasmid using Lipofectamine 2000. Cell lysates were collected at different time points indicated to check ER expression by western blot. Beta-actin was used as an equal loading control. MCF7 cells were used as a positive control. Figure S6 Determination of SELENBP1 expression levels by overexpression or knock-out SELENBP1 plasmids in breast cancer cells. MCF7 cells were transfected with shRNA vector control, or Non-effective 29-mer scrambled shRNA control, or Human SELENBP1-specific shRNA, at 72 h post transfection, cell lysates were collected to proceed for western blot to determine SELENBP1 expression. MDAMB231 cells were transfected with either vector control or SELENBP1 overexpression plasmid. At 48 h of transfection, cell lysates were collected and SELENBP1 expression level was determined by western blot. (PPT)