Journal: Frontiers in Immunology
Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Figure Lengend Snippet: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p
Article Snippet: Human GILZ 3′UTR cDNA was amplified using the Expand High fidelity PCR System (Sigma-Aldrich, # 11732641001) and the following primers: 5′-GCC TAC TAG TGC AGA GCC ACT AAA CTT G-3′ and 5′-AAT AGA GCT CAC TCT CAC AAA ACC CGC TAC-3′.
Techniques: Activation Assay, Affinity Magnetic Separation, Expressing, Western Blot, Quantitative RT-PCR