Lipofectamine transfection agent

Bone marrow derived macrophages (BMDMs) were isolated by flushing femurs of adult mice and culturing the resulting cells in 10% FBS 1% Pen/Strep-containing DMEM supplemented with 10ng/mL recombinant m-CSF (Peprotech) for 7 days. 5μg of immunogenic HT DNA (In vivogen) was complexed with a Lipofectamine transfection agent (ThermoFisher) at a ratio of 1:1.5 in serum free-media and added to 1 million BMDMs in a 6-well multi-well plate with serum- and mCSF-containing media for each experiment. Two independent experiments were performed, yielding n = 5 for each condition. For inhibition of secondary signaling via the IFNAR receptor, cells were treated with 20μg/ml of MAR1-5A3 IFNAR neutralizing antibody or isotype control (BioXCell).

We purchased the following siRNAs from Invitrogen: Silencer Select Negative Control No. 1 siRNA, ZNF277 SILEE SELECT SIRNA ASSAY ID S22065, and CTNNB1 SILEER SIRNA ASSAY ID 146154. For siRNA transfection experiments, human colon cancer cells and HEK293 cells were seeded in 6- and 96-well plates at approximately 10% confluence and incubated at 37^oC for 24 hours. siRNA duplex oligos targeting ZNF277 or nontargeting control oligos were transfected into cells with Lipofectamine Transfection Agent (Thermo Fisher Scientific) according to the manufacturer’s instructions. One to 2 days following transfections, cells in 96-well plates were used for cell proliferation assay, and cells in the 6-well plates were harvested for immunoblotting to confirm and quantify siRNA knockdown.