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. 2020 Sep 24;117(41):25679–25689. doi: 10.1073/pnas.2005111117

Fig. 3.

Fig. 3.

The 15-LO-oxidized lysoPLs are TLR4-activating ligands. SynEVs composed of PLs and lysoPLs were prepared. (A, C, E, G, I, and K) WT or TLR4 KO BMDMs were stimulated for 6 h with synEVs or oxidized synEVs (10 μg/mL) in A, singlePLs or oxidized single PLs (10 μg/mL) in C, 20:4 lysoPI or oxidized 20:4 lysoPI (5 μg/mL) in E, G, I, and K, or LPS (2 ng/mL) as a positive control. Il1b, Il6, Il23a, Ccl24, and Tnfaip6 mRNA levels were determined using qPCR. (B) HEK293T cells expressing hTLR4 and hMD-2/CD14 as well as firefly luciferase under NF-κB promotor and Renilla luciferase for normalization were stimulated with oxidized single PLs (5 μg/mL) or LPS (10 ng/mL) as a positive control for 24 h. Negative controls are transfected but unstimulated cells. A dual luciferase test for NF-κB activity was performed. (D) The 15-LO oxidation products of 20:4 lysoPI that were determined by MS. (F, H, and J) WT or TLR4 KO BMDMs were stimulated for 16 h with LPS (2 ng/mL), 20:4 lysoPI, or oxidized 20:4 lysoPI (10 μg/mL). Il-6 and Ccl24 concentrations in supernatants or Il-23 production in cell lysate were measured by ELISA. ND, not detected. (L and M) WT or TLR4 KO BMDCs were stimulated for 16 or 8 h to detect Il-6 and Il-23, respectively, with LPS (2 ng/mL), stressEVs (10 μg/mL), 20:4 lysoPI, or oxidized 20:4 lysoPI (10 μg/mL). Il-6 and Il-23 concentrations in supernatants were measured by ELISA. (A, C, and K) Three indep. exps. (n = 3); t test (two-tailed; paired or one-tailed; paired for KO); (B) three indep. exps. (n = 6 or 9); t test (two-tailed; unequal var.); n.s., not significant; (E and G) three indep. exps. (n = 4); t test (two-tailed; paired or one-tailed; paired for KO); (I) two indep. exps. (n = 2); t test (two-tailed; paired or one-tailed; paired for KO); (F) two indep. exps. (n = 5); t test (two-tailed; unequal var.); (H) two indep. exps. (n = 4); t test (two-tailed; unequal var. or one-tailed; unequal var. for KO); (J) two indep. exps. (n = 3); t test (two-tailed; unequal var.); and (L and M) two indep. exps. (n = 6); t test (two-tailed; unequal var. or one-tailed; unequal var. for KO).