Figure 6. Glucocorticoid or AAT inhibition of NFκB activation, inhibition of IL-8 production, and induction of angiopoietin-like 4 is glucocorticoid receptor-dependent.

(A) Western blot of whole-cell lysates of THP-1^control and THP-1^GR-KD macrophages for GR and β-actin. Densitometry of the GR band on immunoblot normalized for β-actin (***p<0.001 compared to Scr-shRNA lentivirus). The immunoblot and densitometry shown are representative and the mean of three independent experiments, respectively. (B) RNA sequencing (RNAseq) for the GR gene (NR3C1) transcript of the THP-1^control and THP-1^GR-KD cells using shRNA-lentivirus technology (***p<0.001 compared to Scr-shRNA lentivirus). (C) THP-1^control and THP-1^GR-KD macrophages were left untreated or pre-treated with cortisol, AAT, or both at the indicated concentrations for 30 minutes, and after stimulation with lipopolysaccharide (LPS) for 6 hours, p65-NFκB binding assay to its consensus oligonucleotide was performed. THP-1^control and THP-1^GR-KD macrophages were left untreated or pre-treated with cortisol, AAT, or both at the indicated concentrations for 30 minutes, and after stimulation with LPS for (D) 6 hours or (E) 24 hours, the supernatants were assayed for IL-8 by ELISA. (F) Glucocorticoid (cortisol or dexamethasone) or AAT induction of ANGPTL4 in THP-1^control and THP-1^GR-KD macrophages. Experiments in (C) / (F) and (D) / (E) are the mean ± SEM of three and four independent experiments, respectively, with each experiment done in duplicates. *p<0.05, **p<0.01, ***p<0.001. Blue bars=control shRNA (THP-1^control), red bars=GR shRNA (THP-1^GR-KD). NS=not significant, KD=knockdown, LV=lentivirus, Scr=scrambled.