p53 loss impairs glucocorticoid repression of NF-κB target genes. (A) NF-κB luciferase assay in THP-1 cells transduced with a GFP control (Left) or shp53–GFP (Right). Cells were treated for 6 h with 10 ng/mL TNF ± 1 μM Dex. ***P < 0.001. (B) NF-κB luciferase assay in WT (Left) and p53KO MEF cells (Right) following 24-h treatment with 10 ng/mL TNF ± 1 μM Dex. **P < 0.01. (C) NF-κB luciferase assay in p53KO MEF cells (Left) and in p53KO MEF cells reconstituted with WT p53 (Right) following a 6-h treatment with 10 ng/mL TNF ± 1 μM Dex. ***P < 0.001. (D) qPCR analysis measuring mRNA levels of NF-κB target genes, Cox2 (Left), and MCP-1 (Right) in BMDMs treated with 10 ng/mL LPS ± 1 μM Dex for 2 h. mRNA levels normalized to cyclophilin A.

p53 loss does not alter upstream GR signaling but does impair Dex-mediated transcription. (A) Immunofluorescence of endogenous GR localization in WT (Left) and p53KO (Right) BMDMs treated with or without 1 μM Dex for 30 min. (B) qPCR analysis measuring mRNA levels of GR target genes, Mt2 (Left) and Fkbp5 (Right) in BMDMs treated with or without 1 μM Dex for 2 h. (C) Time course showing GILZ mRNA levels measured by qPCR in BMDMs isolated from WT and p53KO mice and treated with or without Dex for each time point as indicated. mRNA levels normalized to cyclophilin A.

p53 identified as potential gene of interest by high-throughput screening. (A) Schematic representation of screen design. siRNA oligos prearrayed into 384-well plates, were reverse transfected into 293T cells stably transfected with a NF-κB luciferase reporter for 48 h. Cells were then treated with 10 ng/mL TNF ±1 μM Dex in duplicate for 24 h. (B) KEGG analysis. A total of 290 genes of interest were analyzed to determine reactions and/or pathways that were statistically overrepresented. A total of 16 of 29 overrepresented categories with P value <0.05 are shown. (C) Graphical representation of average normalized raw luciferase values of individual wells in the Invitrogen kinase library treated with TNF (y axis) and TNF + Dex (x axis). Red points indicate 10 wells containing sip53. Raw values in each 384-well plate were normalized to that plate's average luciferase activity value. (D) NF-κB luciferase assay in 293T cells transfected with sip53, siGR, si luciferase, sip65, or a siRNA negative control for 48 h and treated with 10 ng/mL TNF ± Dex for 24 h. **P < 0.01.

p53 loss does not alter the upstream NF-κB signaling cascade. (A) Western blot analysis measuring total protein levels of IκBα (Top) and phosphorylated IκBα (Middle). p65 total protein levels (Bottom) are shown to indicate loading control. WT MEF cells (Left) and p53KO MEF cells (Right) were treated for 10, 20, 30, or 60 min with 10 ng/mL TNF ± 1 μM Dex as indicated. (B) Immunofluorescence of endogenous p65 expression in WT (Left) and p53KO (Right) BMDMs treated with 10 ng/mL TNF ± 1 μM Dex for 30 min. (C) Immunofluorescence of endogenous p65 phosphorylation at serine 311 in WT (Left) and p53KO (Right) BMDMs treated with 10 ng/mL TNF ± 1 μM Dex for 30 min. (D) Immunofluorescence of endogenous p65 phosphorylation at serine 276 in WT (Left) and p53KO (Right) BMDMs treated with 10 ng/mL TNF ± 1 μM Dex for 30 min. NT, no treatment; TNF + D, TNF + Dex.

p53 loss impairs glucocorticoid rescue of death in a mouse model of LPS shock. (A) Kaplan–Meier survival curve analysis measuring death from LPS shock. WT and p53KO C57BL6 mice treated with 50 mg/kg LPS ± 10 mg/kg Dex by i.p. injection are shown. (B) Temperature analysis of WT and p53KO mice treated with LPS. (C) Temperature analysis of WT and p53KO mice treated with LPS + Dex. Measurements recorded every 6 h over a 48-h period and every 24 h up to 5 d posttreatment using a rectal thermometer probe. (D) Proposed mechanisms of p53 role in GR repression of NF-κB. p53 may play a direct or indirect role in GR and p65 complex formation in the nucleus (Left) and/or in GR transcriptional activity (Right).