Journal: Proceedings of the National Academy of Sciences

Article Title: Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages

doi: 10.1073/pnas.2406492121

Figure Lengend Snippet: Fig. 2. Osbp mediates macrophage inflammatory responses to 25HC. (A) Secretion of Il6 measured by ELISA in BMDMs left untreated or treated with the indicated Osbp ligands and then stimulated with LPS or PAM3 for 18 h. (B) BMDMs were left untreated or treated with 25HC, itraconazole, or OSW-1 then were left unstimulated or stimulated with LPS and their transcriptomes analyzed by RNA-seq. Top panels: Comparison of the effect of the indicated Osbp ligands on the expression of genes whose expression is altered by at least two-fold (FDR < 0.01; avg. log2(counts-per-million reads) > 2 across all conditions) by treatment with at least one of the indicated Osbp ligands. Bottom panels: The core LPS response was defined as the 637 genes whose expression at 6 h was altered by at least 10-fold (FDR < 0.01; avg. log2(CPM reads) > 2 across all conditions). The plots compare the effect of itraconazole or OSW-1 to that of 25HC on the expression at 18 h following stimulation of core response genes whose expression at 18 h was altered by at least two-fold by either of the indicated treatments on each plot. (C) Percentage of unedited sequences remaining in the indicated cell pools as measured by TIDE sequencing (29). (D) Western blot for Osbp in macrophages differentiated from Osbp g1, g5, and g6 pools. (E) Il6 protein level 18 h following LPS stimulation of macrophages derived from the indicated CIM lines and treated with the indicated compounds. Bars/points indicate the mean of three biological replicates; error bars indicate SEM (A and E). The blue line indicates best fit and r = Pearson correlation (B). Similar results were obtained in 3 (A) or 2 (E) independent experiments.

Article Snippet: 121 No. 33 e2406492121 https://doi.org/10.1073/pnas.2406492121 7 of 8 After blocking with 5% milk, membranes were probed with the relevant primary antibodies (Cleaved Il1b – E7V2A (Cell Signaling #63124), Osbp Polyclonal (Proteintech #11096- 1- AP)) followed by detection with an HRP- conjugated secondary antibody (Anti- mouse IgG- HRP (Cell Signaling #7076), Anti- rabbit IgG HRP (Cell Signaling #7074)).

Techniques: Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison, Expressing, Sequencing, Western Blot, Derivative Assay