Supplementary Figure 6: Analysis of responses of Pld4^–/– DCs, Pld3^–/– thioglycolate-elicited macrophages and PLD3^–/– human cell lines to TLR9 agonists.

(a-c) Responses of Pld4^–/– DCs and Pld3^–/– thioglycolate-elicited macrophages to 2216 fragments when complexed with Lyovec. Triplicate cultures of pDCs, CD8+ DCs and thioglycolate-elicited macrophages of the indicated genotypes were stimulated as in Fig 5 b-e except that the ligands were precomplexed with Lyovec at a concentration of 1 μg/ml. (a) CD8+ DCs; (b) pDCs, (c) macrophages. Mean and standard deviation are shown. This is representative of five experiments (a and b) and three experiments (c). Statistics were determined using an unpaired two-tailed Student’s T test assuming equal variance. (d) TLR9 responses of PLD3-deficient human cells to phosphorothioate (PS) or phosphodiester form of CpG-B (2006) or CpG-A (2216). PLD3 was knocked out in HEK-Blue^hTLR9 cells using CRISPR/Cas9, then individual, sequence-verified clones were assessed for TLR9 responses to the indicated ODNs. NFκB reporter activation was then measured. ODNs were used at 1 μM. Cells were plated in duplicates. Experiment performed three times with similar results. Statistics were determined using a two-tailed Student’s T test assuming equal variance comparing parental HEK-Blue^hTLR9 cells with PLD3KO.10. Mean and standard deviation are shown. (e) PLD3-deficient HEK-Blue^hTLR9 cells as in (d) stimulated with 2216 PS subfragments. Experiment was performed three times with similar results. Statistics were determined using a two-tailed Student’s T test assuming equal variance comparing parental HEK-Blue^hTLR9 cells with PLD3KO.10. Mean and standard deviation are shown. (f) IFN-α or IL-12p70 responses of Pld3^+/– or Pld3^–/– FLT3L-cultured DCs to listed TLR9 stimuli. Cells were plated in triplicates. Experiment was performed once. Mean and standard deviation are shown.