Pgl4.17 basic vector

Normal breast epithelial cell line MCF-10A, breast cancer cell lines MDA-MB-231, MCF-7, SK-BR3, T47D, and Cal51 were obtained from ATCC (American Type Culture Collection, Manassas, USA). MCF-10A was cultured in MCF-10A cell-specific medium (Procell, China). MCF-7, T47D and MDA-MB-231 were cultured in DMEM (Gibco, USA), while Cal51 in 1640 (Gibco, USA). All medium included 10% fetal bovine serum (FBS, NEWZERUM, Australia) and 1% penicillin/streptomycin (Gibco, USA). The cells were kept at 37 °C in a humidified 5% CO₂ atmosphere cell incubator.
The CMTM7 and TCF3 plasmids and their corresponding vectors, as well as miR-182-5p mimic, inhibitor, and the siRNA kits of CMTM7, were obtained from RiboBio (Guangzhou, China). The miRNAs and siRNAs oligonucleotides are listed in Additional file 1: Table S1. The CMTM7 3′UTR region, the miR-182-5p promoter region, and their mutants were cloned into the pGL4.17-basic vector (Promega, USA). The transfection was conducted utilizing Lipofectamine 3000 (Invitrogen, USA) in accordance with the manufacturer’s recommendations. Lentiviruses (RiboBio, China) were utilized to infect MCF-7 cells and generate stable CMTM7 overexpressed cells by the manufacturer’s protocol.

The promoter regions of CMTM7 and miR-182-5p were cloned into a pGL4.17-basic vector (Promega) and transfected into breast cancer cells along with their corresponding controls and PRL-TK plasmid (Promega). Similarly, TOP/FOP-Flash (Genechem) was co-transfected into cells with CMTM7 overexpression or silence vector. The Dual-Luciferase Reporter Assay Kit (Transgene) was utilized to detect Firefly and Renilla luciferase activities in accordance with instructions.

Oligonucleotide primers (nucleotides −1133 to −1107 and −1 to −24 based on previously published sequence information for the upstream region of the ESR1 were used to generate ESR1 promoter fragments from normal placental DNA by polymerase chain reaction (PCR) (Castles et al., 1997 (link)). A 1133 bp (promoter A) of ESR1 promoter expression vector (ERP) was created by cloning this PCR-generated product into the XhoI-HindIII sites of the promoterless luciferase reporter plasmid pGL4.17 basic respectively (Promega, Madison, WI, U.S.A.). Transfections of individual wells were performed using luciferase reporter plasmid (ERP or pGL4.17 basic vector alone), and pRL-TK Renilla luciferase control constructs as a correction for transfection efficiency, and also transfected with pcDNA3.1-RSPO1 overexpression (RSPO1-OE) plasmid (from 0.5 μg/ml to 4 μg/ml)) Cells were then harvested, the dual luciferase assays were performed using a commercial kit (Promega; E1910), Results are shown as fold activity over control activity of the promoterless pGL4.17 basic vector in each set of experiments. All transfections and assays were performed in duplicate with n ≥ 3 individual experiments. GPCR compound library (Selleckchem L2200) was used to for screening of inhibitors that suppress ESR1 upregulation by RSPO1. In each experiment, ESR1-lucieferase reporter cells were treated with RSPO1 for 36–48 hr.