Journal: International immunopharmacology

Article Title: Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

doi: 10.1016/j.intimp.2023.110574

Figure Lengend Snippet: Fig. 3. p53 was required for heat stress-induced necroptosis in the intestine of mice. A. Schematic of the in vitro experimental design for downregulation of p53 in IEC cells before they were subjected to heat stress. B. Effects of the treatment of siRNA-Tp53 on the expression of p53, p-p53, RIP3 and p-RIP3 in IEC cells following heat stress. C. Heat stress-induced upregulation of inflammatory cytokines in IEC cells were restored by knockdown of p53. D. Schematic of the in vivo experimental design for the treatment of Pifithrin-α. In brief, mice were received a daily intraperitoneal (i.p.) injection with Pifithrin-α (3 mg/kg body weight) for three days before they were subjected to heat stress. E and F. Effects of the treatment of Pifithrin-α on the expression of p53, p-p53, RIP3 and p-RIP3 in the intestine (E) and the levels of inflammatory cytokines in plasma of heatstroke mice following heat stress (F) (N = 6 for each group). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: siRNA-Tp53 (sc-29436), siRNA-Ripk3 (sc-61483), siRNA-FITC (sc36869), Tp53 OE (sc-423509-LAC), Ripk3 OE (sc-425224-LAC), Tlr3 OE (sc-431258-LAC), Control lentiviral activation particles (sc-437282) and copGFP control lentiviral particles (sc-108084) were purchased from Santa Cruz Biotechnology, Inc. (USA).

Techniques: In Vitro, Expressing, Knockdown, In Vivo, Injection, Clinical Proteomics

Journal: International immunopharmacology

Article Title: Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

doi: 10.1016/j.intimp.2023.110574

Figure Lengend Snippet: Fig. 4. Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF3-RIP3 pathway was dependent on p53.Tp53-/- mice. B. Effect of p53 deletion on the levels of inflammatory cytokine in the plasma of mice (N = 6 for each group). C. Heat stress-induced upregulation of RIP3, p-RIP3, p-Mlkl and reduction of p- Drp1 were normalized by p53 deletion (N = 6 for each group). D. Effect of p53 deletion on the expression of TLR3 and TRIF in the intestine of mice (N = 6 for each group). E. Schematic of the in vitro experimental design. IEC Tp53-/- cells were treated with Tp53 OE to re-express p53 or with Tlr3 OE to upregulate the expression of TLR3 before they were subjected to heat stress. F. Effect of re-expression of p53 and upregulation of TLR3 on IEC Tp53-/- cells to response to heat stress. G. Immunoblotting analysis showed that heat stress-induced activation of Mlkl was blocked by the deletion of p53, which was activated again by re-expression of p53. H. Flow cytometry with Annexin V-FITC/PI staining was used to evaluate the induction of HS-induced cell death. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: siRNA-Tp53 (sc-29436), siRNA-Ripk3 (sc-61483), siRNA-FITC (sc36869), Tp53 OE (sc-423509-LAC), Ripk3 OE (sc-425224-LAC), Tlr3 OE (sc-431258-LAC), Control lentiviral activation particles (sc-437282) and copGFP control lentiviral particles (sc-108084) were purchased from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Clinical Proteomics, Expressing, In Vitro, Western Blot, Activation Assay, Flow Cytometry, Staining

Journal: International immunopharmacology

Article Title: Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

doi: 10.1016/j.intimp.2023.110574

Figure Lengend Snippet: Fig. 5. Schematic diagram of p53 meditated in heat-induced intestinal epithelial cells. Heat stress promoted the phosphorylation of p53, which further upregulated TLR3 and enhanced the interaction of TRIF-RIP3, leading to the activation of RIP3-MLKL mediated necroptosis in intestinal epithelial cells. The necroptosis of intestinal epithelial cells amplificated the inflammatory of intestine. In turn, inflammation further aggravate the intestine injury.

Article Snippet: siRNA-Tp53 (sc-29436), siRNA-Ripk3 (sc-61483), siRNA-FITC (sc36869), Tp53 OE (sc-423509-LAC), Ripk3 OE (sc-425224-LAC), Tlr3 OE (sc-431258-LAC), Control lentiviral activation particles (sc-437282) and copGFP control lentiviral particles (sc-108084) were purchased from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Phospho-proteomics, Activation Assay

Journal: American Journal of Cancer Research

Article Title: Wild-type TP53 defined gamma-secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs

doi:

Figure Lengend Snippet: Identification of GSI-sensitive GSCs. A. Three RO-sensitive and seven RO-resistant GSCs were identified using sphere formation assay. B, C. The basal expression of NOTCH1 signaling (NOTCH1 and NICD1) and p53 target genes (p21 and BAX) was detected by Western blot analysis. D. GSCs’ sensitivity to three GSIs (RO, avagacestat, and crenigacestat) were tested, and four GSI-sensitive GSCs were identified using sphere formation assay. TP53 status (W: wild-type, M: mutant) is also shown. Data are represented as means ± SD. *: P < 0.05. **: P < 0.01. n = 3.

Article Snippet: Transfection and viral infection For the CRISPR-Cas9-mediated p53 knockout, p53 double nickase plasmid (sc-416469-NIC, Santa Cruz Biotechnology, Dallas, TX) and control double nickase plasmid (sc-437281, Santa Cruz Biotechnology) were transfected into GSC23 with Lipofectamine 2000 (Invitrogen) for 48 hours.

Techniques: Tube Formation Assay, Expressing, Western Blot, Mutagenesis

Journal: American Journal of Cancer Research

Article Title: Wild-type TP53 defined gamma-secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs

doi:

Figure Lengend Snippet: Summary of GSI sensitivity signature. A. Seven of eight wt-p53 GSCs were GSI sensitive, while all mut-p53 GSCs were GSI resistant. B. GSI-sensitive GSCs harbored significantly higher p53 target genes (P21 and BAX) expression than GSI-resistant GSCs. C. GSIs inhibited the sphere formation ability of GSCs harboring wt-p53 instead of mut-p53. D. Wt-p53 GSCs harbored significantly higher NOTCH1 expression than mut-p53 GSCs. Data are represented as means ± SD. **: P < 0.01. n = 3.

Article Snippet: Transfection and viral infection For the CRISPR-Cas9-mediated p53 knockout, p53 double nickase plasmid (sc-416469-NIC, Santa Cruz Biotechnology, Dallas, TX) and control double nickase plasmid (sc-437281, Santa Cruz Biotechnology) were transfected into GSC23 with Lipofectamine 2000 (Invitrogen) for 48 hours.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Wild-type TP53 defined gamma-secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs

doi:

Figure Lengend Snippet: Wt-p53 did not regulate NOTCH1 signaling in GSCs. A. TP53 mutant was knocked out (KO) from GSC23. Then Wt-p53 was introduced into two p53KO subclones. Wt-p53 was also introduced into GSC293, which harbored innate wt-p53. The expression of p53 and NOTCH1 pathway components was detected by Western blot analysis. B. The expression of p53 and NOTCH1 pathway components was detected after Nutlin-3 treatment (24 hours) in two wt-p53 GSCs (GSC34 and GSC293) by Western blot analysis. n = 3. Blue font: wt-p53 cells. Red font: mut-p53 cells.

Article Snippet: Transfection and viral infection For the CRISPR-Cas9-mediated p53 knockout, p53 double nickase plasmid (sc-416469-NIC, Santa Cruz Biotechnology, Dallas, TX) and control double nickase plasmid (sc-437281, Santa Cruz Biotechnology) were transfected into GSC23 with Lipofectamine 2000 (Invitrogen) for 48 hours.

Techniques: Mutagenesis, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: Wild-type TP53 defined gamma-secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs

doi:

Figure Lengend Snippet: NOTCH1 signaling positively regulated wt-p53 activity in GSCs. A. Introducing NICD1 in GSC293 (wt-p53) upregulated p53 and p21 expression. GSCs were treated with doxycycline (2 μg/mL) for 96 hours. B-D. RO-R subclones were derived from GSC34 (wt-p53). B. Both RO-R subclones showed resistance to avagacestat and crenigacestat compared with parental GSC34, as demonstrated using the sphere formation assay. C. The expression of NOTCH1 target genes (HES1 and HES5) and p53 target genes (P21 and BAX) in RO-R subclones was detected by qPCR. D. The expression of NOTCH1 and p53 pathway components in RO-R subclones was detected by Western blot analysis. Data are represented as means ± SD. *: P < 0.05. **: P < 0.01. n = 3. Blue font: wt-p53 cells. Red font: mut-p53 cells.

Article Snippet: Transfection and viral infection For the CRISPR-Cas9-mediated p53 knockout, p53 double nickase plasmid (sc-416469-NIC, Santa Cruz Biotechnology, Dallas, TX) and control double nickase plasmid (sc-437281, Santa Cruz Biotechnology) were transfected into GSC23 with Lipofectamine 2000 (Invitrogen) for 48 hours.

Techniques: Activity Assay, Expressing, Derivative Assay, Tube Formation Assay, Western Blot

Journal: American Journal of Cancer Research

Article Title: Wild-type TP53 defined gamma-secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs

doi:

Figure Lengend Snippet: Doxorubicin synergistically augmented cytotoxicity of GSIs in wt-p53 GSCs. A. GSCs were treated with the combination of GSI and doxorubicin (DXR) for 5 days. The combination effect was analyzed using the Bliss independency model. GSI plus DXR exerted the more apparent synergistic effect in wt-p53 GSCs than in mut-p53 GSCs. B. Combining GSI with DXR exerted more significantly synergistic anti-proliferative effect in wt-p53 GSCs than in mut-p53 GSCs. Each point represents the average combination effect by GSIs and DXR treatment for each cell line. C, D. RO- and DXR-induced apoptosis was tested by flow cytometry. The combination effect was analyzed using the Bliss independency model. RO plus DXR induced more apoptosis in wt-p53 GSCs than in mut-p53 GSCs. See also Figure S5. E. RO- and DXR-induced apoptosis was detected by PARP1 and caspase-3 cleavage levels. RO plus DXR induced PARP1 and caspase-3 cleavage in wt-p53 GSCs. See also Figure S6. Doxorubicin concentration for apoptosis detection was determined by IC50 for each GSC. ***: P < 0.001. n = 3. Blue font: wt-p53 cells. Red font: mut-p53 cells.

Article Snippet: Transfection and viral infection For the CRISPR-Cas9-mediated p53 knockout, p53 double nickase plasmid (sc-416469-NIC, Santa Cruz Biotechnology, Dallas, TX) and control double nickase plasmid (sc-437281, Santa Cruz Biotechnology) were transfected into GSC23 with Lipofectamine 2000 (Invitrogen) for 48 hours.

Techniques: Flow Cytometry, Concentration Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21 Cip1/Waf1 Pathway

doi: 10.1155/2016/7524308

Figure Lengend Snippet: The detection of senescence-associated phenotype in BMSCs at the 1st (P1) and 2nd (P2) and 3rd passage (P3). (a) Representative image of β -galactosidase (SA- β -gal) staining in BMSCs at P1, P2, and P3. (b) Percentage of SA- β -gal positive senescent cells in BMSCs at P1, P2, and P3. (c) and (d) Expression levels of p53 and p21 Cip1/Waf1 proteins in BMSCs at P1 and P3. Data are expressed as mean ± SD, n = 3 for each group, ∗ P < 0.05, ∗∗ P < 0.01 versus P1.

Article Snippet: To investigate whether the p53 pathway was involved in the antisenescence effects of CH in the aging BMSCs, a p53 activator RITA (reactivation of p53 and induction of tumor cell apoptosis; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to evaluate the role of p53 in this process.

Techniques: Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21 Cip1/Waf1 Pathway

doi: 10.1155/2016/7524308

Figure Lengend Snippet: Effects of CH on expression of p53, p21 Cip1/Waf1 , and LC3 proteins. (a) and (b), Expression of p53 protein in the P3 or in the P1 with H 2 O 2 (25 μ M, 6 h). n = 3 for each group, ∗ P < 0.05, ∗∗ P < 0.01 versus Ctl group; # P < 0.05, ## P < 0.01 versus H 2 O 2 group. (c) and (d) Expression of p21 Cip1/Waf1 proteins in the P3 or in the P1 cells treated with H 2 O 2 . n = 3 for each group, ∗ P < 0.05, ∗∗ P < 0.01 versus Ctl group; # P < 0.05, ## P < 0.01 versus H 2 O 2 group. (e) Expression of LC3 protein in BMSCs with or without different concentrations of CH. n = 3 for each group, ∗∗ P < 0.01 versus P1 group; # P < 0.05, ## P < 0.01 versus P3.

Article Snippet: To investigate whether the p53 pathway was involved in the antisenescence effects of CH in the aging BMSCs, a p53 activator RITA (reactivation of p53 and induction of tumor cell apoptosis; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to evaluate the role of p53 in this process.

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21 Cip1/Waf1 Pathway

doi: 10.1155/2016/7524308

Figure Lengend Snippet: Activity of SA- β -gal in BMSCs with RITA (p53 activator) or 3-MA (autophagy inhibitor). (a) Representative image of SA- β -gal staining in the P3 BMSCs treated with RITA a p53 activator (1.0 μ M), CH (15 μ g/mL), or RITA + CH. (b) Percentage of SA- β -gal positive cells in different groups shown in (a). n = 3 for each group, ∗∗ P < 0.01 versus Ctl group; ## P < 0.01 versus RITA group. (c) Representative image of SA- β -gal staining in the P3 BMSCs treated with 3-MA an autophagy inhibitor (5.0 mM), CH (15 μ g/mL), or 3-MA + CH. (d) Percentage of SA- β -gal positive cells in different groups shown in (c). n = 3 for each group, ∗∗ P < 0.01 versus Ctl group; ## P < 0.01 versus 3-MA group.

Article Snippet: To investigate whether the p53 pathway was involved in the antisenescence effects of CH in the aging BMSCs, a p53 activator RITA (reactivation of p53 and induction of tumor cell apoptosis; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to evaluate the role of p53 in this process.

Techniques: Activity Assay, Staining

Journal: PLoS ONE

Article Title: Unraveling Regulatory Programs for NF-kappaB, p53 and MicroRNAs in Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0073656

Figure Lengend Snippet: A, Overlapped target genes of NF-κB and p53. B, Gene regulatory programs of seven TFs (NF-κB, p53, AP1, CEBPB, EGR1, SP1, and STAT3). Genes in underlined, bold and bold-underlined refer to known targets of NF-κB, p53, and both NF-κB/p53, respectively. ↓ and ↑ refer to number of predicted target genes differentially over- and under-expressed in the tumor cells, respectively.

Article Snippet: p53 DNA binding activity was quantitatively assessed using a modified version of the TransAM p53 ELISA kit from Active Motif (Carlsbad, CA).

Techniques:

Journal: PLoS ONE

Article Title: Unraveling Regulatory Programs for NF-kappaB, p53 and MicroRNAs in Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0073656

Figure Lengend Snippet: Enriched signal pathways in the gene regulatory programs of NF-κB and p53.

Article Snippet: p53 DNA binding activity was quantitatively assessed using a modified version of the TransAM p53 ELISA kit from Active Motif (Carlsbad, CA).

Techniques:

Journal: PLoS ONE

Article Title: Unraveling Regulatory Programs for NF-kappaB, p53 and MicroRNAs in Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0073656

Figure Lengend Snippet: A, network of the wt p53-deficient cells. B, network of the mt p53 cells. Every node represents a common target gene of NF-κB, p53, mir21 or mir34ac, and was annotated according to inflammatory and immune responses (green nodes), apoptosis (blue), angiogenesis (yellow), proliferation (red), adhesion (gold), proteolysis (light red) and other processes (light blue). The networks were presented by cytoscape.

Article Snippet: p53 DNA binding activity was quantitatively assessed using a modified version of the TransAM p53 ELISA kit from Active Motif (Carlsbad, CA).

Techniques:

Journal: PLoS ONE

Article Title: Unraveling Regulatory Programs for NF-kappaB, p53 and MicroRNAs in Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0073656

Figure Lengend Snippet: A, a network of hypopharyngeal cancer. B, a network of oral cancer. Every node represents a common target gene of NF-κB, p53, mir21 or mir34ac, and was annotated to inflammatory and immune responses (green nodes), apoptosis (blue), angiogenesis (yellow), proliferation (red), adhesion (gold), proteolysis (light red) and other processes (light blue). The networks were presented by cytoscape.

Article Snippet: p53 DNA binding activity was quantitatively assessed using a modified version of the TransAM p53 ELISA kit from Active Motif (Carlsbad, CA).

Techniques:

Journal: PLoS ONE

Article Title: Unraveling Regulatory Programs for NF-kappaB, p53 and MicroRNAs in Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0073656

Figure Lengend Snippet: UM-SCC 22B (A, the mt p53) and 1 (B, the wt p53-deficient) cells were transfected with siRNA to RelA or TP53 for 48 h or 72 h. Total RNA was isolated, and genes selected from TF-miRNA networks in the were analyzed by real time qRT-PCR. The data were calculated as the mean plus standard deviation from triplicates with normalization by 18S ribosome RNA, and presented as the comparison with the cultured cells transfected with the control siRNA oligos. The open bar represents the cells transfected with control siRNA, and the values have been normalized to 1. The fold changes of the target genes were presented when compared with those transfected with control siRNA. The black bar represents cells transfected with siRNA targeting TP53, while the blue bar represents cells transfected with siRNA targeting NF-κB subunit, RelA/p65. * refers to statistical significance (t test, P <0.05).

Article Snippet: p53 DNA binding activity was quantitatively assessed using a modified version of the TransAM p53 ELISA kit from Active Motif (Carlsbad, CA).

Techniques: Transfection, Isolation, Quantitative RT-PCR, Standard Deviation, Comparison, Cell Culture, Control

Journal: PLoS ONE

Article Title: Unraveling Regulatory Programs for NF-kappaB, p53 and MicroRNAs in Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0073656

Figure Lengend Snippet: Nuclear extract from UM-SCC 1 cells were isolated, and the binding activity was examined on the biotin labeled oligos containing promoter sequences with p53 binding sites. The binding activity of nuclear extract of MCF-7 cells with a biotinylated oligonucleotide containing a known p53 binding site in the CDKN1A gene promoter (“MCF-7 p21 oligo”) was served as a positive control. Specificity of binding in the UM-SCC cells was also tested using cold competition (unbiotinylated) oligonucleotides which contained the wild-type p53 consensus binding sequence (“wt competition”) or mutant DNA sequence of p53 binding site (“mt competition”).The mean and standard deviation of each binding was calculated from triplicate, and the presented data are from one representative of repeated experiments. *refers to statistical significance when p53 binding activity was competed by the mt oligo, to confirm the binding specificity (t test, P <0.05).

Article Snippet: p53 DNA binding activity was quantitatively assessed using a modified version of the TransAM p53 ELISA kit from Active Motif (Carlsbad, CA).

Techniques: Isolation, Binding Assay, Activity Assay, Labeling, Positive Control, Sequencing, Mutagenesis, Standard Deviation

Journal: PLoS ONE

Article Title: Comparing Two Intestinal Porcine Epithelial Cell Lines (IPECs): Morphological Differentiation, Function and Metabolism

doi: 10.1371/journal.pone.0132323

Figure Lengend Snippet: qPCR—used primer sequences.

Article Snippet: For the analysis of p53 cells were treated with p53 activator (10 μM; Merck, Darmstadt, Germany) for 24 h.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Comparing Two Intestinal Porcine Epithelial Cell Lines (IPECs): Morphological Differentiation, Function and Metabolism

doi: 10.1371/journal.pone.0132323

Figure Lengend Snippet: Comparison of microarray data and qPCR. Important genes of the significantly regulated pathways shown in Table 2 were analysed via qPCR. The microarry data are shown and two reference genes were used to illustrate significant differences between both cell lines. Asterisks indicate significant differences from IPEC-1.

Article Snippet: For the analysis of p53 cells were treated with p53 activator (10 μM; Merck, Darmstadt, Germany) for 24 h.

Techniques: Comparison, Microarray

Journal: PLoS ONE

Article Title: Comparing Two Intestinal Porcine Epithelial Cell Lines (IPECs): Morphological Differentiation, Function and Metabolism

doi: 10.1371/journal.pone.0132323

Figure Lengend Snippet: The expression of p53 was analysed by microarray, qPCR and Western blot. (A) IPEC-J2 showed a 27.4 fold increase of the p53-mRNA level in comparison to IPEC-1. This was confirmed by qPCR and a 300-fold increase was detected in IPEC-J2. On the other hand, no protein expression was observed in IPEC-1/IPEC-J2 and Caco-2 as well as in the cells, which were treated with the p53-activator. In the next step, the expression of BAX, BAD and BCL-X was analysed using microarray, qPCR and Western blot. (B) BAD showed a 1.8 fold increase in IPEC-J2 in the microarray. Same results were found in qPCR, but no differences in the protein expression were found between the cell lines. A significant BCL-X up-regulation was detected in the microarray in IPEC-J2 but no differences were present between IPEC-1 and IPEC-J2 in the Western blot analyses. No BAX-protein was observed in both cell lines. When the total RNA and protein content was examined, significant differences were found between the cell lines. IPEC-J2 showed a higher RNA- and protein content in comparison to IPEC-1. RPL10A as important gene of the 60S ribosomal subunit was significantly decreased in the microarray but not in qPCR in IPEC-J2 in comparison to IPEC-1. Western blot analyses showed a slightly decrease of the protein in IPEC-J2 over time. (C) The RNA and protein content per cell was measured in both cell lines. IPEC-J2 showed a significant higher level in RNA and proteins. On the other hand, a slight decrease on protein level of RPL10A was found in IPEC-J2 in comparison to IPEC-1 in Western blot analyses.

Article Snippet: For the analysis of p53 cells were treated with p53 activator (10 μM; Merck, Darmstadt, Germany) for 24 h.

Techniques: Expressing, Microarray, Western Blot, Comparison

Journal: Oncotarget

Article Title: TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells

doi:

Figure Lengend Snippet: A. TUNEL staining on LNCaP cells. Green fluorescence represents apoptotic cells and Blue fluorescence represents DAPI. B. The 2064 bp 5′-flanking region of the human trpm8 gene with ARE I and ARE II sites were analyzed further for the presence of possible putative p53 binding sites using TRANSFAC ® 7.0. The authenticity of the TFBS was confirmed by other promoter binding software tools; TFBIND, AlGGEN server PROMO (Version 3.0) and Transcription Element Search Software (TESS). The WebLogo 3.4 software was used to generate the sequence logo for p53 binding site (1399 bp upstream of the transcription start site) on trpm8 gene promoter. The error bars indicate an approximate, Bayesian 95% confidence interval. C. Reverse-transcription PCR analysis of control and p53 overexpressing LNCaP cells (primers are listed in ). D. EMSA was performed with 2 μg of nuclear extract (NE) from control and treated cells. Panel; 1 is for the p53 (3) EMSA probe set in the absence and presence of control NE provided in the kit. Panel; 2 The authenticity of the p53 binding site identified on the TRPM8 promoter was confirmed by performing EMSA using biotinylated probes . Panel; 3 shows TRPM8 promoter probe EMSA in the presence of anti-p53 antibody. E. Monitoring p53 activation in the NE using TransAM, assay Kit (Active Motif) in the presence and absence of competing TRPM8 promoter probe. The results are graphically represented as bar diagrams.

Article Snippet: The p53 activation in nuclear extracts was monitored using p53 (3) - EMSA (Affymetrix, Santa Clara, CA) and ELISA-based TransAM™ p53 kit (Active Motif) according to the manufacturer's protocol (see SMethods).

Techniques: TUNEL Assay, Staining, Fluorescence, Binding Assay, Software, Sequencing, Activation Assay