Lipojet in vitro transfection kit

The specific Silencer Select small interfering RNAs (siRNAs) targeting human INTS11 and a corresponding negative control siRNA were purchased from Thermo Fisher Scientific (Assays s29893, s29894, s29895, AM4635). The specific siRNA targeting human NACK and a corresponding negative control siRNA were purchased from Santa Cruz Biotechnology (sc-77433, sc-37007). A LipoJet In Vitro Transfection Kit was purchased from SignaGen Laboratories. The day before transfection, cells were seeded in cell culture plates containing complete RPMI medium and incubated for 24 h. The following day, transfection was performed using siRNAs and the LipoJet In Vitro Transfection Kit according to the manufacturer's instructions. The final concentrations of INTS11 and NACK siRNAs were 60 nM and 100 nM, respectively. The cells were collected either 72 h or 96 h after transfection.
pcDNA plasmids encoding human Notch1 (0.1 µg), Maml1 (0.01 µg), or NACK (0.01–0.1 µg) were used to transfect HEK293T cells. Transfection was performed as described for EAC cells with the following modifications. Complete DMEM medium was used for HEK293T cells and cell were collected 48 h after transfection.

FLO-1 and OE33 cells were transfected with control siRNA or NRF2 specific siRNA using the LipoJet In Vitro Transfection Kit (SignaGen Laboratories, Frederick, MD, USA) according to the manufacturer’s protocols. Forty-eight hours after infection, cells were seeded in the density of 1000 cells/well in the 6-well plates and cultured at 37 °C for another two weeks. For the clonogenic survival assay, tumor cells were seeded in 6-well plates at the density of 1000 cells per well. The next day, the cells were treated with Brusatol (30 nM), CDDP (3 µM), or a combination of Brusatol (30 nM) and CDDP (3 µM) for 24 h, followed by the removal of the media and replacement of full media for two weeks. Each experiment was set in triplicate. Cells were stained with 0.5% crystal violet solution. The images of the plates were analyzed using ImageJ software (version 1.53k, National Institutes of Health, Bethesda, MD, USA) and statistically analyzed using Prism 9 software (version 9.3.1, GraphPad Software, San Diego, CA, USA).

Ultra-pure LPS from Escherichia coli O111:B4 was from Sigma-Aldrich. LG100268 and MLN7243 was from Cayman. Nigericin, muramyl dipeptide (MDP), Flagellin, poly(dA:dT) and heat-killed P. aeruginosa (HKPA) were from Invivogen. ATP, cycloheximide (CHX), MG132, and Bafilomycin A1 were from Sigma-Aldrich. CytoTox 96® Non-Radioactive Cytotoxicity LDH Assay was from Promega. LipoJet™ In Vitro Transfection Kit was from SignaGen Laboratories.

The dog fetal fibroblast cells were maintained in complete cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, CA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) Glutamax (Life Technologies), 1% (v/v) non-essential amino acids (Life Technologies), 1% (v/v) antibiotic-antimycotics (Life Technologies), and 0.1% (v/v) 2-mercaptoethanol (Life Technologies). The incubation conditions for the primary culture were 37°C and 5% CO₂ in a humid incubator.
Transfection of each linearized plasmid vector into canine fibroblasts was performed using LipoJet^™ In Vitro Transfection Kit (SignaGen Laboratories, MD, USA) according to the manufacturer’s instructions. EGFP expression after transfection was monitored by IncuCyte ZOOM^™ (Essen BioScience, MI, USA) and average green object intensity per image was calculated using IncuCyte ZOOM^™ software.