Figure 4
From: Phosphorylated STAT5 regulates p53 expression via BRCA1/BARD1-NPM1 and MDM2
STAT5 phosphorylation downregulates NPM1 by impairing the BRCA1-BARD1 ubiquitin ligase. (a) 2.5 × 105 TF-1 cells maintained in GM-CSF supplemented medium were transiently transfected with pGL3-luc-NPM1 promoter vector on day 1 using Lipofectamine 2000 and were deprived of GM-CSF on day 2 for 16 h. The GM-CSF-deprived TF-1 cells were treated with or without IL-3 (100 ng/ml) for 2 h on day 3. Luciferase assays were performed for each experimental condition. (b) Quantitative reverse transcriptase-PCR were performed to analyze the relative NPM1 mRNA levels in HEK 293T cells transfected with either RFP-STAT5A vector or backbone control for 48 h. (c) 1 × 106 TF-1cells were deprived of GM-CSF and simultaneously treated with TAME hydrochloride (5 mM) for 16 h, followed by 3 h of IL-3 treatment (100 ng/ml). (d) 1 × 106 TF-1 cells deprived of GM-CSF overnight were treated with 10 μM ALLN for 1 h prior to IL-3 treatment (100 ng/ml) for 3 h. (e) HeLa cells were plated on T-75 culture flasks on day 0 and co-transfected with pcDNA3-FLAG-NPM1 and pcMV-(HA-ubiquitin)4 vectors on day 1. The transfected cells were treated with either hEGF (100 ng/ml) alone for 2 h or inhibitor 573108 (225 μM) for 1 h prior to a 3 h hEGF treatment. Cells receiving different treatments were lysed and immunoprecipitated with mouse monoclonal anti-NPM1 antibody (5 μg per condition) and probed with anti-HA antibody to determine the ubiquitin conjugated to NPM1 in each condition. (f) 1 × 106 TF-1 cells deprived of GM-CSF overnight were stimulated with IL-3 at the concentration of 100 ng/ml for 3 h. The cell lysate were subjected to immunoblotting assays to determine the expression levels of BRCA1, BARD1 and NPM1. (g) HEK 293T cells (6 × 105 per well) were plated in a six-well plate on day 0 and transfected with either RFP-STAT5A or RFP-backbone vectors on day 1. The transfected cells were harvested and lysed for immunoblotting assays on the expression levels of BRCA1, BARD1 and NPM1. (h) HEK 293T cells (6 × 105 per well) were plated in a six-well plate on day 0 and transfected with either RFP-STAT5A or RFP-backbone vectors for 6 h, followed by a 1.5 h incubation with ALLN at a concentration of 10 μM. The cells were harvested and lysed on day 2 for immunoblotting assays on the changes in expression level of BRCA1 and BARD1. For all experiments, statistical analysis was based on three independent assays (mean±S.D.). For immunoblotting, one out of the three representative experiments was shown each time
