Extended Data Fig. 2: nLDL and macrophage TLR responses quality control.
From: LDL delivery of microbial small RNAs drives atherosclerosis through macrophage TLR8
(a) Total protein (top) and neutral lipid (bottom) of ox LDL, bovine serum albumin (BSA), and 10 human nLDL of independent donors resolved by agarose gel electrophoresis. Image represents two independent experiments. (b) TBARS assay of nLDL samples, or matched LDL samples treated with copper sulfate as indicated (limit of detection = 0.625 μM) (n = 10 independent preparations). (c) Quantification of total protein and lipids of DGUC-VLDL, -LDL, -HDL, or BSA following fractionation with 2x-Superose-6 columns. Lipoprotein data are matched to a single donor representative of >10 independent experiments. (d) mRNA expression of primary human macrophages (CD14 + ; GM-CSF/IFNγ) pre-treated with C29 (200 μM) for 30 min, then stimulated with PAM3CSK4 (2 ng/mL) for 4 h (n = 3 biological replicates(BR)). (e) Normalized NF-κB-driven luciferase activity of HEK293T cells over expressing an empty vector, hTLR7 or hTLR8 following treatments with vehicle (Ctr, n = 4 BR), nLDL (0.5 mg/ml, n = 4 BR), ssRNA40 (TLR8 ligand; 2 μg/mL, n = 4 BR), R848 (TLR7 ligand; 10 μM, n = 3 BR), or CL075 (TLR8 ligand; 2.5 μg/mL, n = 3 BR). (f-h) mRNA expression of THP-1 macrophages electroporated with siRNA against TLR7,TLR8, or no siRNA (Control, n = 4 BR) and then treated with (f) nLDL (0.5 mg/mL, n = 6 BR), (g) R848 (10 μM, n = 6 BR) or (h) ssRNA40 (0.5 μg/mL, n = 6 BR). (i) Relative NF-κB-driven luciferase activity of HEK293T cells over expressing an empty vector (n = 7–8), mTLR7 (n = 7–8) or mTLR8 (n = 6-8) treated with mock transfection, R848 (10 μM), ssRNA40 (2 μg/mL), or nLDL (0.5 mg/ml) for 24 h. (j) mRNA expression in wild-type (WT) and Tlr7-/- BMDMs following treatment with 0.5 mg/ml nLDL, or 1 μg/mL ssRNA40 for 24 h (n = 3 BR). (k) Relative NF-κB-driven luciferase activity of HEK293T cells over expressing human or mTLR8 pre-treated with CU-CPT9a and exposed to nLDL for 24 h (n = 4 BR). Data are mean ± SEM. (d) Two-way ANOVA; Sidak’s multiple comparisons test, ***p < 0.001, ****p < 0.0001. (e, i-k) Two-way ANOVA; Benjamini, Krieger and Yekutieli FDR (Q = 0.05), *q < 0.05, **q < 0.01, ***q < 0.001). (f-h) One-way ANOVA; Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Numerical source data, statistics, exact p values and q values are provided.
