Pgl3 firefly luciferase reporter vector

The promoter sequence of YB-1 target gene was cloned into pGL3 firefly luciferase reporter vector (Promega, USA) with sequence-specific primers (Table 1). Then, the recombinant pGL3 was co-transfected into melanoma stem cells or breast cancer stem cells with pRL-TK renilla luciferase reporter vector (Promega, USA). The activities of firefly luciferase of pGL3 and renilla luciferase of pRL-TK were determined following the dual-luciferase reporter assay protocol recommended by Promega. The cells were washed with PBS and then lysed with passive lysis buffer (Promega, USA). Subsequently, the cell lysate was subjected to the detection of firefly luciferase activity (M1), followed by the examination of renilla luciferase activity (M2). The promoter activity was calculated according to the following formula: Promoter Activity = M1/M2.

The human CDC20B promoter was cloned into the pGL3 Firefly Luciferase reporter vector (Promega) with SacI and NheI cloning sites. The promoter sequenced ranged from −1073 to +104 relative to the transcription start site. 37.5 ng of pGL3 plasmid were applied per well. pCMV6-Neg, pCMV6-E2F1 (NM_005225) and pCMV6-E2F4 (NM_001950) constructs were from Origene. 37.5 ng of each plasmid was applied per well. 25 ng per well of pRL-CMV (Promega) was applied in the transfection mix for transfection normalization (Renilla luciferase). HEK 293T cells were seeded at 20,000 cells per well on 96-well plates. The following day, cells were transfected with the indicated plasmids (100 ng of total DNA) with lipofectamine 3000 (Invitrogen). After 24 h, cells were processed with the DualGlo kit (Promega) and luciferase activity was recorded on a plate reader.

Transfection and luciferase assays were performed as previously described [31 (link),32 (link)]. First, plasmid DNA was amplified by transformation of Library Efficiency DH5α competent cells (Thermo Fisher Scientific). A pGL3 Firefly Luciferase Reporter Vector (Promega, Madison, WI, USA) containing glucocorticoid-responsive test promoter pMMTV-DNA [17 (link)] and a Renilla Luciferase Control Reporter Vector (Promega) were purified using the innuPREP Plasmid Mini Kit 2.0 (AJ Innuscreen, Berlin, Germany). The amount of extracted vector was determined using a NanoDrop 2000 instrument (Thermo Fisher Scientific). Next, cerebEND cells were co-transfected with both vectors using the Lipofectamine 3000 Reagent Kit (Thermo Fisher Scientific). MMTV transcription activity was measured using the Dual-Luciferase Reporter Assay System (Promega) by determining the firefly luciferase signal in a Lumat LB9507 (Berthold Technologies) and normalizing it to transfection efficiency by determining the Renilla luciferase activity.

The promoter sequence of the YB-1 gene was cloned into the pGL3 firefly luciferase reporter vector (Promega, USA) with sequence-specific primers (Supplementary Table 1). Then, the recombinant pGL3 vector was cotransfected into MCF-7 breast cancer stem cells with the pRL-TK Renilla luciferase reporter vector (Promega, USA). The firefly luciferase activity of pGL3 and Renilla luciferase activity of pRL-TK were measured in accordance with the dual luciferase reporter assay protocol recommended by Promega. Cells were washed with PBS and lysed in passive lysis buffer (Promega, USA) for 30 min. Subsequently, the cell lysate was subjected to detection of firefly luciferase activity (M1), followed by detection of Renilla luciferase activity (M2). The promoter activity was calculated according to the following formula: Promoter Activity=M1/M2.
We also examined the effect of ERα expression on YB-1 promoter activity. The ERα protein-coding gene sequence was cloned into the pcDNA3.1 plasmid (Invitrogen, USA). Then, cells were transfected with 150 nM recombinant plasmid using Lipofectamine 2000 (Invitrogen, USA).

Genomic DNA was purified from MCF-7 cells using a Wizard^® SV Genomic DNA Purification System (Promega: A2360). A construct containing 250 bp upstream of the start site of MUC16 transcription (hereafter refer to as 250 bp promoter construct) was amplified by genomic PCR. This PCR product was cloned into pCR 2.1 TOPO (Life Technologies; K456001). This fragment was ligated into the promoter less pGL3 firefly luciferase reporter vector (Promega; E1751). The primers used were Fwd: 5′-AGAGAGAGAGAGAGAGAGGATCATT-3′ Rev 5′-AATGATCCTCTCTCTCTCTCTCTCT-3′. Site directed mutagenesis was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies Inc; 210518) according to the manufacturer's instructions.

HEK293T cells were co-transfected with pGL3 firefly luciferase reporter vector (Promega) containing a series of LCE1C promoter region (region #1, #2, #3, or #4), pRL Renilla luciferase internal control vector (Promega), and pCMV6myc mammalian expression vector containing a series of TAp63 (6Myc-tagged TAp63-WT, -AA, or –DD) using PLUS Reagent and Lipofectamine Reagent (Invitrogen). After transfection, the cells were incubated for 48 h, and the luciferase activity was assayed using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacture’s instruction. The results were normalized against Renilla luciferase activity. SV40 promoter was used as a positive control of luciferase assay. All data are shown as means ± s.d. (standard deviation) from three independent experiments.

COS-7 cells were transfected with the pcDNA3 expression vector (Invitrogen) containing the coding region of wild type, G145C, or S351–354dup SOX11; the pGL3 Firefly Luciferase reporter vector (Promega) containing the GDF5 core promoter was a kind gift from Akinori Kan (Harvard Medical School, Boston, MA) [80] (link); and the pRL-TK vector (Promega) containing Renilla luciferase driven by a ubiquitous tyrosine kinase promoter to control for transfection efficiency. Transfections were performed using Fugene 6 (Promega), following manufacturer's instructions. The total mass of DNA and molar ratios of pGL3 and pRL-TK were held constant across transfections, which were repeated a minimum of 6 times. Dose response curves were generated using wild type SOX11 at 0∶100, 1∶20, 1∶10, and 1∶5 molar ratios to the GDF5 reporter. The mutant SOX11 variants were transfected at a 1∶5 molar ratio to the GDF5 reporter. Firefly and Renilla luciferase activity were measured 24–36 hours post transfection using the DualGlo Luciferase Assay System (Promega). Data was analyzed as follows: Firefly luciferase (FFLuc) was baselined against untransfected control (UTC) samples ( = FFLuc – UTC) and normalized using the Renilla luciferase (RLuc). The Relative Luciferase Activity (RLA) was calculated as (FFLuc-UTC)/RLuc and compared between experimental and control transfections.