Apoc3

LNPs and Apos were incubated in ddH₂O water overnight at 4 °C. LNPs equivalent of 5 µg encapsulated plasmid DNA were incubated with 3 µg of each of the different human Apos; ApoA1 (Sigma, SRP4693), ApoB (Sigma, 178456), ApoC3 (Sigma, A3106), ApoD (Sigma, SRP4326), ApoE2 (Peprotech, 350-12), ApoE4 (Peprotech, 350-04) and ApoH (Sigma, G9173), unless otherwise stated, in a final volume of 200 µl. For control experiments (i.e. LNP only), LNPs were incubated with ddH₂O water (in the absence of any apolipoprotein) overnight at 4 °C in a final volume of 200 µl. For Cryo TEM reactions were set up as described above in the absence of ddH₂O.

Cell viability reagent WST-1 was purchased from Roche Diagnostics, Almere, The Netherlands. Human serum and human plasma purified lipoproteins (VLDL, LDL, HDL) and apolipoproteins (ApoA, ApoB, ApoC1, ApoC2, ApoC3) were all purchased from Sigma-Aldrich. Human lipoprotein deficient serum was from Merck, Amsterdam, the Netherlands. Heat-killed Staphylococcus aureus (HKSA), Escherichia coli (HKEB), Salmonella typhimurium (HKST) were all purchased from Invivogen, Toulouse, France. Ultrapure LPS derived from E. coli K12 and purified LTA from Staphylococcus aureus (both from Invivogen, Toulouse, France) were used at the indicated concentrations. Synthetic bacterial lipopeptides Pam₃CSK₄, Pam₂CSK₄, FSL-1 (all from EMC microcollections, Tübingen, Germany) were used at the indicated concentrations. Non-phagocytic HEK293 TLR2-TLR6, HEK293 TLR4 stable transfectants and HEK293 TLR null control cells were purchased from Invivogen, Toulouse, France. HEK293 TLR2-TLR6 and HEK293 TLR null transfectants were stably transfected with the NFκB reporter plasmid pNiFty2-Luc, HEK293 TLR4 cells contained the NFκB reporter pNiFty2-SEAP (Invivogen, Toulouse, France). Cells were maintained in DMEM (Invitrogen) supplemented 10 % FBS, 4.5 g/L glucose and the appropriate antibiotics according to the manufacturer’s protocols.