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. 2015 Nov 6;16(12):1656–1663. doi: 10.15252/embr.201540861

Figure EV3. TLR7 mRNA knockdown in k.o. THP‐1 cells and consequent responsiveness, Sa12 derivative sensitivity, bacterial infection‐driven type I IFN or IL‐6 production and inhibition of PBMCs or whole blood, respectively, and further species PBMC responsiveness.

Figure EV3

  1. Knockdown upon transfection of control (scramble) or two different TLR7 mRNA‐specific siRNAs (siTLR7‐1/3) in and cytokine release of respective 3‐day differentiated THP‐1 cells upon challenge (Rel., relative; accum., accumulation; Loxo, loxoribine; R848, small molecule, 50 μg/ml; tRNA, 200 ng/well of 96‐well plate E. coli transfer RNA; n = 3).
  2. Sequence alignments of Sa12 and Sa12s6U with another Sa12 variant carrying in addition, as compared to the latter, ORN and, reminiscent of the respective motif in HsmtD1, a UGA motif (blue, core sequence; red, A6U; underlined, A7G). Diagram depicts PBMC activities upon challenges with Sa12 and derivatives (n = 3).
  3. PBMC type I IFN production upon pretreatments (T2.5, TLR2‐neutralizing mAb; chloroquine, lysososmal function inhibitor) and challenge with TLR ligands or infection (5 × 104 cfu/ml; n = 2; corresponding to Fig 4B).
  4. Cytokine release of whole blood culture upon lysosome inhibition and challenge with heat‐inactivated (hi) and viable bacteria (triangle, decreasing dose of infection; n = 3; corresponding to Fig 4C).
  5. Cytokine release of Sus (S.) scrofa and Macaca (M.) mulatta PBMCs upon challenge with ORN variants (n = 3).
Data information: Graphs show mean ± SD; *P ≤ 0.05; **P ≤ 0.01; unpaired t‐test; –, unchallenged; n.d., not detected; P3C, Pam3CSK4.