Viafecttm transfection reagent

The 293T cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). High-sugar DMEM (Hyclone, Logan, UT, USA), fetal bovine serum (Gibco, Waltham, MA, USA), and 1% streptomycin/penicillin (Invitrogen, Waltham, MA, USA) were used. Plasmids were transfected into cells using ViaFectTMTransfection Reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

BSR T7/5 cells were seeded in 6-well plates to reach a confluency of approximately 75% at the time of transfection. Transfection of plasmid DNA with ViaFect^TM Transfection Reagent (Promega) was performed as described above. After an incubation period of 24 h the cells were washed with ice-cold PBS following lysis with RIPA buffer (Millipore, Burlington, MA, USA) and protease inhibitors (Roche, Basel, Switzerland). Cells were scraped and incubated at 4 °C for 30 min and centrifuged (13,000× g, 10 min, 4 °C). Lysates were mixed 1:1 with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Reducing agent β-mercaptoethanol was added, for analysis under reducing conditions. Samples were treated with PNGase F (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. After boiling the mixtures, the cell lysates were loaded and separated on 4–20% or any kD polyacrylamide gels (Bio-Rad) and transferred to a Immobilon-P transfer membrane (Millipore). RSV F proteins were visualized with palivizumab and HRP-conjugated goat anti-human IgG. Monoclonal anti β-actin antibody (Sigma, Tokyo, Japan) to detect β-actin levels served as loading control. Chemiluminescence was measured after incubation with a chemiluminescent substrate (Thermo Fisher Scientific) using a GenoPlex Chemi camera (VWR, Radnor, PA, USA).

pNL3.1 (#N1031, Promega) was selected as vector and pGL4.53 (#E5011, Promega) as control. Mutant and Wildtype fragment DNAs were inserted into pNL3.1 vector by Quick Ligation Protocol (M2200, New England Biolabs) using NheI-HF (R3131S, New England Biolabs) and HindII-HF (R3104S, New England Biolabs) according to manufacturer instructions (see Supplementary Table S10 for fragment sequences). Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions. Transfection was done by ViaFect^TM Transfection Reagent (E4981, Promega) according to manufacturer instructions with medium to final volume ratio of 4:1. Cells were assay after 24 hours using Nano-Glo Dual-Luciferase Reporter Assay System (N1610, Promega) according to instructions with CentroXS3 LB960 (Berthold Technology) and measurement time of 1 second for both ONE-Glo and NanoDLR.

MDA-MB-231 cell line was cultured in High-glucose Dulbecco’s Modified Eagle’s Medium (Sigma; D6429) supplemented with 10% fetal bovine serum, 1% Penicillin/Streptomycin, and 2 mM L-Glutamine. pcDNA3-PRB was a gift from Elizabeth Wilson (Addgene plasmid # 89130; http://n2t.net/addgene:89130; RRID: Addgene_89130) (178).
MDA-MB-231 cells were transfected with 1 µg of pcDNA-PRB plasmid construct for 24 hours using ViaFect^TM Transfection reagent (Promega; E4982) according to manufacturers' instruction. Negative control with prepared by treating the cells with plasmid-free ViaFect reagent. RNA was extracted from cell pellets using PureLink RNA Mini kit (Invitrogen; 12183018A), as per the manufacturer’s instructions. RNA samples were cleaned up from potential DNA contaminations using TURBO DNA-free TM Kit (Invitrogen; AM1907).

The mammalian two-hybrid (M2H) assay was performed on human cervical carcinoma cell (HeLa cells, kindly provided by Stem Cell Bank, Chinese Academy of Sciences) using the Checkmate^TM M2H system (Promega, United States) in accordance with the manufacturer’s instructions. Briefly, gene fragments encoding SCY2 mature peptide and scyreprocin were cloned in frame with pACT vector and pBIND vector (primer sequences were listed in Supplementary Table 1), respectively. HeLa cells were maintained in minimal essential medium (Invitrogen, United States) supplemented with 10% fetal bovine serum (FBS; Gibco). Cells were plated at ∼5.0 × 10⁴ cells well^–1 on a 48-well cell culture plate (Thermo Fisher). Vector combinations, including (1) pACT/pBIND/pG5luc, (2) pACT-SCY2/pBIND/pG5luc, (3) pACT/pBIND-scyreprocin/pG5luc, and (4) pACT-SCY2/pBIND-scyreprocin/pG5luc, were transiently transfected into HeLa cells using the ViaFect^TM Transfection Reagent (Promega) according to the manufacturer’s instructions. Cells were harvested and lysed in passive lysis buffer (Promega) at 48-h post-transfection. Reporter activities were measured using the Dual-Luciferase Reporter Assay System (Promega) on a GloMax 20/20 luminometer (Promega). Experiments were performed in triplicate.

For detection of RORα/γ activity, CRC cells cultured in 96-well plates were transfected with a pGL4.20 construct containing the RORE motif using ViaFect^TM Transfection Reagent (Promega, E4892). After transfection, cells were treated with 10 μM atorvastatin (MedChemExpress (MCE), HY-B0589, Monmouth Junction, USA), conditioned medium containing LPD-FBS or an exogenous LDL supplement (40 μg/mL). For detection of NEDD4 promoter activity, we cloned the wild-type NEDD4 promoter region (−2500 bp to +500 bp upstream of the transcription start site) and mutant promoter region (with deletion of the two binding site sequences) into the pGL4.20 plasmid. CRC cells were transfected with the RORα or RORγ plasmid, the wild-type or mutant NEDD4 promoter plasmid and the Renilla luciferase vector using ViaFect^TM Transfection Reagent. The luciferase activities were measured with a Dual-Glo^® Luciferase Assay System (Promega, E2920) after 36 h of transfection, as described previously [56 (link)]. For MYC overexpression, pcDNA3.1-MYC plasmid or the corresponding empty vector were transfected into CRC cells using ViaFect^TM Transfection Reagent. All the plasmids and constructs were synthesized by Umine Biotechnology Co., LTD (Guangzhou, China).