Lipofectamine ltx kit

P-gp activity was determined by measuring the intracellular accumulation of Rho123 in A549 taxol-resistant human lung cancer cells (KeyGen Biotech, Nanjing, China), and a well-known P-gp inhibitor, 10 μM verapamil (Sigma-Aldrich, St. Louis, MO, USA), was used as the positive control. The U-87 MG cell lines were plated in 6-well plates and transfected with 2 μg ABCB1 plasmid with Lipofectamine^® LTX kit (Thermo Fisher Scientific, Waltham, MA, USA) for 8 h. Cells were pre-treated with the 2 HM formulations for 4 h at 37 °C before incubation with 5 μg/mL Rho123 at 37 °C for 1 h. The Rho123 accumulation assay was terminated by washing the cells with ice-cold PBS (Invitrogen, Carlsbad, CA, USA) and re-suspending them in 100 μL PBS. The intracellular Rho123 signal was detected with flow cytometry (FACSAria™ III flow cytometer; BD Biosciences, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Data were analyzed in FlowJo v10.6.1 software (BD Biosciences, CA, USA) and are expressed as a percentage of Rho123 positive cells.

We used two different ISRE-luciferase reporter assays: (i) an assay using the ISRE3 reporter plasmid (pGL4.10[luc2] backbone, Promega #E6651), which contains, as previously described^{158 (link)}, three repeats of the ISRE sequence (5’-GGAAAGGGAAACCGAAACTGAA-3’) separated by spacers designed on the basis of the ISRE motif from the ISG15 promoter, (ii) an assay using the ISRE5 reporter plasmid, which contains, as previously described^{139 (link)}, five repeats of the ISRE sequence 5’-GGGAAAGTGAAACTA-3’. HEK293T cells were transiently transfected, in 96-well plates, with the (ISRE) reporter plasmid (100 ng/well and 100 μL DMEM-10% FBS medium), the pRL-SV40 vector (Promega, #E2231, 40 ng/well) and the IRF1 WT or mutant p.CMV6 plasmid (100 ng/well), with the Lipofectamine LTX kit (Thermo Fisher Scientific, #15338–100), according to the manufacturer’s instructions. Cells were used for the ISRE assay with the Dual-Luciferase system kit (Promega #E1980), according to the manufacturer’s protocol, 24 hours after transfection. Signal intensity was determined with a Victor X4 plate reader (Perkin Elmer). Experiments were performed in triplicate, and dual reporter activity is expressed as the fold-induction relative to cells transfected with the empty vector.

We used the ISRE3 reporter plasmid (pGL4. 10[luc2] backbone, Promega #E6651), as previously described [25 (link)]. HEK293T cells in 96-well plates were transiently transfected with the (ISRE) reporter plasmid (100 ng/well and 100 µL of DMEM-10% FBS medium) and the pRL-SV40 plasmid (Promega, #E2231, 40 ng/well), with or without the IRF1 WT p.CMV6 plasmid (50 ng/well), with or without IRF8-WT or mutant forms, in the presence of the Lipofectamine LTX kit (Thermo Fisher Scientific, #15,338–100) and with or without EV, to ensure that the same amount of plasmid was present in each well, in accordance with the manufacturer’s instructions. Cells were used for the ISRE assay with the Dual-Luciferase system kit (Promega #E1980), according to the manufacturer’s protocol, 24 h after transfection. Signal intensity was determined with a Victor X4 plate reader (Perkin Elmer). Experiments were performed in triplicate, and results are expressed as dual reporter activity.

Empty vector (EV) and a plasmid containing the DDK-tagged IRF1 cDNA were obtained from a commercial source (#RCPS100001 and #RC203500, respectively, Origene). Constructs carrying single-nucleotide mutant alleles were generated from this plasmid by mutagenesis with appropriate primers, with the Pfu Ultra II Fusion HS DNA (#600674, Agilent) polymerase, followed by digestion with DpnI (#R0176L, New England Biolab). For assessments of the reinitiation of translation, methionine codons were mutated to alanine codons (ATG>GCG). Plasmids were amplified in competent E. coli cells (#C3019H, New England Biolab) and purified with a maxiprep kit (#12663, Qiagen). HEK293T cells were transiently transfected with the various constructs at a concentration of 2.5 μg/mL, with the Lipofectamine LTX kit (#15338100, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Retroviral plasmids and vectors were prepared as previously described, with primers for site-directed mutagenesis or deletion, and were produced in Phoenix cells^{157 (link)}.

For transfection experiments, CGNs were cultured at density of 40 000 cells per coverslip. Neurons were transfected with either pcDNA3, full-length p75^wt, p75^NFkB or p75^{RhoGDI/cell death} plasmids using Lipofectamine LTX kit (Invitrogen, Cat: 15338500) 48 h after plating. 250 ng plasmid per well in the 24-well plate was used.

RNAi-Ready pSIREN-RetroQZsGreen was stored by the College of Animal Science and Technology, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University. The mRNA sequence of MT1 (ACCESSION: JN038179) was used for the target sequence of RNAi. Oligonucleotide sequences for the generation of siRNAs used in this study were designed using the Ambion siRNA Target Finder at the Ambion Inc. web (www.ambion.com/techlib/misc/siRNA_finder.html), sequence synthesis was conducted by Shanghai Sangon Limited (Shanghai, China). The three interference sequences were named shRNA1, shRNA2, and shRNA3 (Table 1). For transient transfection, mesenchymal cells were washed with 1× PBS, transfections were carried out using a Lipofectamine LTX kit (Invitrogen, Eugene, OR, USA) according to the manufacturer’s directions. Desired plasmids were diluted in Opti-MEM medium (Lifetechnology, Waltham, MA, USA), incubated for 5 min and later mixed with LTX for 30 min. Mesenchymal cells were collected for protein extraction or other analyses after transfection.