Journal: British Journal of Pharmacology

Article Title: Transforming growth factor-β impairs glucocorticoid activity in the A549 lung adenocarcinoma cell line

doi: 10.1111/j.1476-5381.2012.01885.x

Figure Lengend Snippet: (A) Immunofluorescence of GRα in A549 cells treated with TGF-β for 24 h and dexamethasone (Dex; 10 nM) for 2 h before fixation. The images were obtained on Leica DMI6000B live cell imaging system at a magnification of 400×. Nuclei are stained blue with DAPI, or GRα is detected with a FITC-labelled secondary antibody. The image in Figure 7A is representative of five similar experiments, grouped data for which is presented in (B). (C) Cells were incubated using an identical protocol to that in Figure 7A except that 30 nM dexamethasone (Dex) was used. At the end of the 2 h dexamethasone incubation, cells were separated into cytosolic (C) and nuclear (N) extracts that were subjected to SDS-PAGE and immunoblotted with anti-GRα. Equal proportions of the whole cell extract representing the nuclear and cytosolic fractions were loaded. The nuclear fraction is identified by enrichment of the nuclear proteins, lamin A and C. Equivalence of cytosolic fraction loading was established by β-actin levels (not shown). (D) Grouped data for GR-α levels in the nuclear fraction, corrected for lamin A and C and expressed as a percentage of the level in vehicle/Dex 30 nM group (n = 4). (E) GRα gene expression is shown 4 h after exposure to dexamethasone (Dex; 30 nM), TGF-β 100 pM or the combination. Data are presented as mean and SEM of 3 experiments (each determined in triplicate). *P < 0.05, significantly different from control gene expression.

Article Snippet: Immunofluorescence Cells seeded at 30 000 cells per chamber in an eight-chamber cell culture slide (Nunc, Roskilde, the Netherlands) were allowed to attach for 24 h then starved in DMEM containing 0.25% BSA for a further 24 h. Cells were then treated with TGF-β (40 pM) for 24 h before the addition of dexamethasone (10 nM) for 2 h. Cells were fixed in 10% vv −1 neutral buffered formalin for 5 min, washed in PBS and incubated with the primary antibody against GRα (GR rabbit polyclonal #sc-1003: Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Immunofluorescence, Live Cell Imaging, Staining, Incubation, SDS Page, Gene Expression, Control

Journal: Chinese Medical Journal

Article Title: Role of p300 in the pathogenesis of Henoch-Schonlein purpura nephritis and as a new target of glucocorticoid therapy in mice

doi: 10.1097/CM9.0000000000000380

Figure Lengend Snippet: Expression of glucocorticoid receptor and resistance gene at mRNA and protein level in each group. (A) Expression of GRα and β, transforming growth factor-β1 and activator protein-1 mRNA in each group. (B, C) Expression of GRα and β, transforming growth factor-β1 and activator protein-1 protein in each group. Glucocorticoid receptor (GR) α expression was significantly higher in Group VI in comparison with Group III ( ∗ P < 0.05); the expression of GRβ did not differ significantly. Meanwhile, p300 knockdown caused a significant increase in the expression of transforming growth factor-β1 and activator protein-1 at the levels of mRNA and protein, which suggests that p300 is involved in the occurrence of glucocorticoid resistance ( ∗ P < 0.05). Group II: Non-knockout model group; Group III: Non-knockout dexamethasone therapy group; Group VI: p300 knockout dexamethasone therapy group.

Article Snippet: The primary antibodies used in this study were as follows: GRα (BOSTER Bio Inc, Wuhan, China), GRβ (Bioss Bio Inc, Beijing, China), TGF-β1 (BOSTER Bio Inc), activator protein (AP)-1 (Bioss Bio Inc), and GAPDH (Santa Cruz Bio Inc, Santa Cruz, USA).

Techniques: Expressing, Comparison, Knockdown, Knock-Out