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1.
FIGURE 2.

FIGURE 2. From: Tumor Protein p63/Nuclear Factor ?B Feedback Loop in Regulation of Cell Death.

Both RelA and TAp63α mediated cell death pathways. A and B, MCF10A cells transfected with the pCMV4-RelA expression cassette (0–1.5 μg) for 24 h. A, immunoblotting with the indicated antibodies. B, MTT assay. C and D, MCF10A cells transfected with the RelA shRNA (0–1.5 μg) for 48 h followed by immunoblotting with the indicated antibodies. C, MCF10A cells were transfected with the 1 μg RelA or p65Res constructs for 24 h followed by transfection with RelA shRNA (0–1.5 μg) for 24 h and immunoblotted with the indicated antibodies. D, MTT assay. *, p ≤ 0.05 (n = 3) compared with control (untransfected cells or scrambled shRNA). Error bars, S.D.

Tanusree Sen, et al. J Biol Chem. 2011 December 16;286(50):43204-43213.
2.
FIGURE 3.

FIGURE 3. From: Tumor Protein p63/Nuclear Factor ?B Feedback Loop in Regulation of Cell Death.

TAp63α induced cell death pathways through RelA. MCF10A cells were transfected with the 20 nm TAp63α siRNA for 24 h followed by the increasing concentration of the ectopic RelA expression cassette (0–1.5 μg) for 24 h. A, immunoblotting with the indicated antibodies. B, MTT assay. MCF10A cells were transfected with the 1 μg of RelA shRNA followed by the TAp63α expression cassette (0–1.5 μg). C, immunoblotting with indicated antibodies. D, MTT assay. For the MTT assay, values from control cells transfected with the empty vector or control (scrambled) siRNA/shRNA were taken as 100%. *, p ≤ 0.05 (n = 3) compared with control (untransfected cells). Error bars, S.D.

Tanusree Sen, et al. J Biol Chem. 2011 December 16;286(50):43204-43213.
3.
FIGURE 1.

FIGURE 1. From: Tumor Protein p63/Nuclear Factor ?B Feedback Loop in Regulation of Cell Death.

TAp63α overexpression and siRNA affect the protein levels of RELA and cell death/cell cycle regulators and cell survival. A and C, immunoblotting of total lysates from MCF10A (A) and H1299 (C) cells with the indicated antibodies. B and D, MTT assay of MCF10A (B) and H1299 (D) cells after transfection with an increasing concentration (0–1.5 μg) of TAp63α for 24 h. E, Immunoblotting of total lysates (MCF10A cells) with the indicated antibodies. F, MTT assay performed on MCF10A cells after siRNA-dependent TAp63α down-regulation (20 nm siRNA, for 48 h). For CASP3 and PARP, positions of molecular mass markers are shown on the left. For the MTT assay, values from control cells transfected with the empty vector or scrambled siRNA were taken as 100%. *, p ≤ 0.05 (n = 3) compared with control (untransfected cells). Error bars, S.D.

Tanusree Sen, et al. J Biol Chem. 2011 December 16;286(50):43204-43213.
4.
FIGURE 6.

FIGURE 6. From: Tumor Protein p63/Nuclear Factor ?B Feedback Loop in Regulation of Cell Death.

TAp63α increased levels of RelA-dependent genes. MCF10A cells were transfected with the TAp63α expression plasmid (0–1.5 μg). A and B, Ready-To-GlowTM Secreted Luciferase assay for the NF-κB/RelA response element-driven Metridia luciferase activity. 12 h after transfection of H1299 (A) and MCF10A (B) cells with the indicated constructs, the medium was replaced, and then after 16 h, samples of the medium were analyzed for secreted Metridia luciferase activity. The -fold induction was calculated following the substrate addition. C and D, luciferase reporter assay for the Cdkn1a (C) and Bbc3 (D) promoters. MCF10A cells were transfected with the basic TA-luc, pCdkn1a-luc, or Bbc3-luc constructs along with the Renilla luciferase plasmid and the TAp63α or RelA or basic vector for 24 h. Immunoblotting for each sample was performed for TAp63α, RelA, and β-actin. Firefly luciferase activity was normalized for the Renilla luciferase activity. Data presented as relative-fold change units (RFU) from the basic TA-luc activity are designated as 1. *, p ≤ 0.001(n = 5) compared with basic TA-luc activity, or pCdkn1a-luc (C) or Bbc3-luc (D) activities alone.

Tanusree Sen, et al. J Biol Chem. 2011 December 16;286(50):43204-43213.
5.
FIGURE 4.

FIGURE 4. From: Tumor Protein p63/Nuclear Factor ?B Feedback Loop in Regulation of Cell Death.

TAp63α-RELA protein interaction modulated the RELA protein level in MCF10A cells. A, complex formation between the TAp63α with RELA proteins. Top left panel, MCF10A cells were transfected with the TAp63α-FLAG or an empty FLAG vector. Immunoprecipitation (IP) was performed using anti-FLAG matrix and blotted with the anti-RELA antibody. Top right panel, MCF10A cells were transfected with the RelA-FLAG cassette or an empty FLAG-vector. Total lysates were precipitated with either control matrix or anti-RelA matrix and blotted with an anti-TP63 antibody. Immunoblotting for TAp63 and β-actin levels was used as input and loading controls, respectively. B, MCF10A cells were transfected with the scrambled siRNA or TAp63α siRNA for 48 h. Cells were treated with the 10 μm proteasome inhibitor, MG-132, for the indicated time periods. Total lysates were immunoblotted with the anti-RELA or anti-p63 antibodies. C–E, MCF10A cells were transfected with the 1.5 μg of RelA expression cassette along with the 1.5 μg of empty vector (C), 1.5 μg of TAp63α expression plasmid (D), or 20 nm TAp63α siRNA (E) for 48 h, as indicated. Cells were then treated with 100 μg/ml cycloheximide (CHX). At the indicated time points, total lysates were analyzed by immunoblotting with indicated antibodies. F, MCF10A cells were co-transfected with the RelA and HA-Ub expression plasmids, without or with the TAp63α expression vector or TAp63α siRNA for 36h and then treated with MG-132 for an additional 10 h. Total lysates were precipitated with anti-HA-matrix and blotted with the anti-RELA antibody. Immunoblot analysis for β-actin showed the loading levels.

Tanusree Sen, et al. J Biol Chem. 2011 December 16;286(50):43204-43213.
6.
FIGURE 5.

FIGURE 5. From: Tumor Protein p63/Nuclear Factor ?B Feedback Loop in Regulation of Cell Death.

TAp63α mediated the RelA transcriptional regulation. MCF10A cells were transfected with the TAp63α expression plasmid (0–1.5 μg). A, immunoblotting of the nuclear and cytosolic fractions with indicated antibodies. B, semi-qRT-PCR analysis for mRNA levels of indicated genes. C, ChIP assay performed with MCF10A cells, 72 h after transfection with the TAp63α expression construct or an empty vector, as well as with the TAp63α siRNA or scrambled siRNA. Total lysates aliquots of the same samples (for specific and nonspecific regions) were tested for both TAp63α and β-actin levels using immunoblotting, as indicated. TAp63α-bound RelA promoter DNA was precipitated with the 4A4 antibody followed by the PCR for the specific (−1900 to −1551bp; supplemental Fig. S1) and nonspecific regions (−600 to −351bp; supplemental Fig. S1), designated as ChIP. PCR with the purified DNA (Input) for both regions is shown. D, MCF10A (left) and H1299 (right) cells were transfected with the basic TA-luc and RelA-luc constructs along with the Renilla luciferase plasmid, with or without the TAp63α and/or RelA expression cassette for 24 h. The firefly luciferase activity was normalized for the Renilla luciferase activity. Immunoblotting for TAp63α and β-actin was performed. Data are presented as relative -fold change units (RFU) from the basic TA-luc activity designated as 1. *, p ≤ 0.001(n = 5) compared with basic TA-luc activity or RelA-luc activity alone.

Tanusree Sen, et al. J Biol Chem. 2011 December 16;286(50):43204-43213.

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