Journal: bioRxiv
Article Title: Estrogen receptor alpha (ERα) driven trans-regulation of mitotic checkpoint complex (MCC) components affects the clinical outcome of breast cancer
doi: 10.1101/2024.08.31.610592
Figure Lengend Snippet: MAD2 and BUB1B are transcriptionally activated by ERα. (A) Schematic representations showing the estrogen response elements (EREs) in the promoter regions of MAD2 and BUB1B . (B) E2 (17β-estradiol) treatment up-regulates MAD2 and BUB1B promoter activity in MCF-7 cells. The top panel shows the map of MAD2 and BUB1B promoter regions used for luciferase reporter assay. pSS-MAD2 construct contains an 1128-bp region (−1098 to +30 nt) of the MAD2 gene and the pSS-BUB1B construct contains a 1546-bp region (−1454 to +92 nt) of the BUB1B gene, cloned into pGL3 basic vector. MCF-7 cells were transiently transfected with pSS-MAD2 (250 ng) and pSS-BUB1B (500 ng), followed by E2 treatment, and proceeded for luciferase reporter assay. **p < 0.005; (two-tailed unpaired Student’s t-test) (C) Ectopic ERα up-regulates MAD2 and BUB1B promoter activity in MDA-MB-231 cells. MDA-MB-231 cells were co-transfected with pSS-MAD2 (250 ng) and pSS-BUB1B (500 ng) along with 1.5 µg of ectopic ERα (pCMV3-N-FLAG-ESR1), followed by E2 treatment and proceeded for luciferase reporter assay and plotted as discussed in (B). *p < 0.05; **p < 0.005; ***p < 0.0005 (two-tailed unpaired Student’s t-test). (D) Deletion of ERE results in a significant decrease in luciferase activity. Map of the two deletion constructs, ΔERE pSS-MAD2, and ΔERE pSS-BUB1B were shown in the top panel. MCF-7 cells were transfected with either ΔERE pSS-MAD2 (250 ng) or ΔERE pSS-BUB1B (500 ng), followed by E2 treatment. Data were plotted as discussed in (B). **p < 0.005; ns, not significant (two-tailed unpaired Student’s t-test). (E) ERα physically recruits to MAD2 and BUB1B promoters. The total cell extract of MCF-7 was prepared and Chromatin immunoprecipitation was done using antibodies specific for ERα and normal IgG as control. Precipitated chromatin was estimated by qRT-PCR. The results are expressed as a percent of input. *p < 0.05; **p < 0.005; (two-tailed unpaired Student’s t-test). (F) Representative scatter plot depicting the Pearson’s correlation analysis between the gene expression values (TPM) of MAD2 and BUB1B , extracted from TCGA BRCA cohort using UCSC Xena database. The Pearson correlation coefficient is reported as the ‘r’ value. ** *p < 0.0005; (two-tailed unpaired Student’s t-test). All the experiments (B-E) were performed in triplicates and error bars were represented as means ± s.d.
Article Snippet: The fragments were then subcloned into luciferase reporter vector pGL3 basic (Promega) using restriction enzyme BglII (New England Biolabs).
Techniques: Activity Assay, Luciferase, Reporter Assay, Construct, Clone Assay, Plasmid Preparation, Transfection, Two Tailed Test, Chromatin Immunoprecipitation, Control, Quantitative RT-PCR, Expressing
