Journal: Cellular and Molecular Neurobiology

Article Title: CREB2 Functions as a Central Mediator of Oxidative Neuronal Death Triggered by Microglial Glutamate Release Under Neuroinflammatory Conditions

doi: 10.1007/s10571-026-01695-w

Figure Lengend Snippet: p53 inhibition suppresses glutamate-induced CREB2 activation and downstream cytotoxic signaling. A HT22 cells were pretreated with 10 µM PFTα (a p53 inhibitor) for 1 h, followed by 5 mM glutamate (Glu) exposure for an additional 24 h. Protein levels of CREB2 and GADD45α were assessed by Western blotting and quantified (left panels). Cell viability was measured using the MTT assay (right panel). B Immunofluorescence staining of CREB2 was performed following the same treatment as in ( A ). Representative images show nuclei stained with Hoechst and CREB2 with FITC (scale bar = 50 μm). C Cells were transfected with p53 siRNA (sip53) for 24 h, followed by 5 mM glutamate treatment for an additional 24 h. Western blotting for CREB2 and p53 and their quantification (left panels) are shown. Cell viability was determined using the MTT assay (right panel). D Cells were transfected with CREB2 siRNA (siCREB2) for 24 h prior to glutamate treatment (5 mM, 24 h). Western blotting was performed to assess expression of phosphorylated p53 (Ser15), CREB2, and GADD45α (left panels). Quantification is shown in the left panel. Cell viability was assessed by MTT assay (right panel). E Cells were transfected with GADD45α siRNA (siGADD45α) for 24 h, followed by glutamate treatment (5 mM, 24 h). Protein levels of CREB2 and GADD45α were analyzed by Western blotting and quantified. Data are presented as mean ± SD ( n = 3). GAPDH served as a loading control. Statistical significance was determined using the following tests: One-way ANOVA with Bonferroni’s post hoc test for MTT in ( A ) [F(3, 8) = 333, p < 0.0001], ( C ) [F(3, 8) = 282, p < 0.0001], and ( D ) [F(3, 8) = 269.6, p < 0.0001]; and Two-way ANOVA with Bonferroni’s post hoc test for WB quantification in ( A ) [F(3, 16) = 437.4, p < 0.0001], ( C ) [F(3, 16) = 12.85, p = 0.0002], ( D ) [F(3, 24) = 83.83, p < 0.0001], and ( E ) [F(3, 16) = 1054, p < 0.0001]. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant

Article Snippet: Membranes were blocked with 5% skim milk in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature and subsequently incubated overnight at 4 °C with primary antibodies against CREB2/ATF4 (mouse monoclonal, clone B-3, 1:100; Santa Cruz Biotechnology, cat. no. sc-390063, RRID: AB_10917316), p53 (1:1000; Cell Signaling Technology (CST), cat. no. 12571, RRID: AB_27113937), phospho-p53 (Ser15) (1:1000; CST, cat. no. 9284, RRID: AB_331464), GADD45α (1:1000; CST, cat. no. 4632, RRID: AB_10694428), GAPDH (1:1000; CST, cat. no. 5174, RRID: AB_10622025), and β-actin (1:200; Santa Cruz Biotechnology, cat. no. sc-47778, RRID: AB_2714189).

Techniques: Inhibition, Activation Assay, Western Blot, MTT Assay, Immunofluorescence, Staining, Transfection, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 1. p53 and WOX1 binding interactions. A, Molt-4 T cells were exposed to UV light (240 mJ/cm2), followed by culturing for 10–120 min. Both cytosolic and nuclear fractions were pre- pared and examined for phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (p-WOX1) and their nuclear translocation. UV light also increased the levels of nuclear histone H3 and its acetylation at lysine 9 (H3k9). By immunoprecipitation (IP) using p53 IgG antibody, UV light was shown to induce binding of cytosolic p53 with WOX1 in Molt-4 T cells.B,weconfirmedtheUVlight-inducednuclear translocation of p53 and WOX1 in Molt-4 cells by immunofluorescence microscopy. Cells were stained with IgG antibodies against WWOX (N19; goat) and full-length p53 (FL-393; rabbit), respec- tively (3, 4). Secondary staining was performed using anti-goat IgG (conjugated with Texas Red) and anti-rabbit IgG (conjugated with Cy2). Nuclei were stained with 4,6-diamidino-2-phenylindole. C, monocytic U937 cells were exposed to UV light (240 mJ/cm2) and shown to have increased phos- phorylation of cytosolic p53 at Ser46 and WOX1 at Tyr33, and p53 binding with p-WOX1. D, similarly, neuroblastoma SK-N-SH cells were exposed to deferroxamine (500 M) for 1 h to induce hypoxic stress and shown to have an increased binding of p53 with WOX1.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Binding Assay, Phospho-proteomics, Translocation Assay, Immunoprecipitation, Immunofluorescence, Microscopy, Staining

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 3. Tyr33-phosphorylated WOX1 binds Ser46-phosphorylated p53 but not Ser46-deleted p53. p53-negative H1299 cells were transfected with DsRed-tagged wild type p53, p53S46, or DsRed alone by electroporation. The cells were cultured for 48 h and examined by fluorescence microscopy. More than 70% of cells expressed DsRed. UV light increased the binding of p-WOX1 with the wild type p53-DsRed, but not p53S46- DsRed, as determined by co-immunoprecipitation using antibodies against p-WOX1 (25). The ectopically expressed proteins are shown (see the bottom panel). IgH, IgG heavy chain.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Transfection, Electroporation, Cell Culture, Fluorescence, Microscopy, Binding Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 2. Mapping of p53 and WOX1 interac- tions by yeast two-hybrid analysis and temper- ature-dependent WOX1 phosphorylation. A, analysis of protein/protein interaction in the cyto- plasm was performed using a Ras rescue-based yeast two-hybrid system (3, 10, 25, 29). In positive controls, binding of WOX1 with p53 and MafB self- interactionareshown,asevidencedbythegrowth of yeast at 37 °C using selective agarose plates containing galactose. In negative controls, no yeast growth at 37 °C was observed for the empty pSos/pMyr vectors and collagenase and lamin C. Human p53 physically interacted with the N-ter- minal WW domains of murine WOX1. Deletion of an N-terminal proline-rich segment (amino acids 66–100; see block No. 2) from the full-length p53 did not abrogate the binding. Similarly, deletion of Thr18 or Ser20 in p53 could not prevent its binding with WOX1. In contrast, deletion of Ser46 in p53 significantly reduced the binding. Alterations of Tyr33 to Arg in the first WW domain of WOX1 abol- ished its binding with p53. p53 structure was as follows: transcriptional activation domain (1); pro- line-rich region (2); DNA-binding domain (3); tet- ramerization domain (4). B, to examine WOX1 phos- phorylation at Tyr33, some yeast cells were grown at room temperature for 2 days in a galactose-contain- ing broth (to reach a preplateau stage), followed by culturing at 37 °C for 1 h to induce the activation of Ras pathway. Heat induced the expression of p53, WOX1, and its phosphorylation at Tyr33 (p-WOX1) in yeastcellstransfectedwithwildtypep53andWOX1. Incontrast,noWOX1phosphorylationwasobserved in cells transfected with p53S46 mutant and wild type WOX1.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Phospho-proteomics, Binding Assay, Blocking Assay, Activation Assay, Expressing, Transfection, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 10. A schematic model for p53 interactions with WOX1 and MDM2. A, UV light induces phosphorylation of p53 at Ser46 and WOX1 at Tyr33 and their complex formation in the presence of MDM2. Nutlin-3 inhibits MDM2 and appears to alter p53 conformation (p53*), thereby stabilizing p53 and its interaction with WOX1. This p53 is not phosphorylated at key serines. UV light restores the complex formation of p-p53-p- WOX1-MDM2. B, WOX1 alone binds MDM2 probably via its C-terminal short-chain alco- hol dehydrogenase/reductase domain, and nutlin-3 dissociates the binding. UV light restores the binding.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Phospho-proteomics, Binding Assay

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: Network pharmacology analysis identifies p53 as a core ferroptosis-related target of FF in UC. ( A ) Venn diagram illustrating the intersection of FF compound targets with ferroptosis- and UC-related targets. ( B ) Protein–protein interaction (PPI) network of the common targets. Node size and color intensity represent the degree of connectivity, with TP53 (p53) identified as the core target. ( C ) Compound-target-pathway network diagram. The inner pink nodes represent the 38 intersecting targets linking FF, UC, and ferroptosis. ( D ) Gene Ontology (GO) enrichment analysis of the common targets, categorized into Biological Process (BP, red), Cellular Component (CC, green), and Molecular Function (MF, blue). ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques:

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: FF modulates the expression of ferroptosis-related proteins in colon tissue via the p53 pathway. ( A ) Representative immunohistochemical (IHC) images of p53, SLC7A11, and GPX4 expression in colon sections (scale bar = 50 μm). ( B – D ) Quantitative analysis of the relative protein expression levels of p53 (B), SLC7A11 (C), and GPX4 (D). Data are presented as the mean ± SD ( n = 3 independent experiments). ### p < 0.001 versus the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the DSS model group.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 5 PAI-1 induced p53 upregulation is critical for the subsequent maladaptive response. A, Western blot assessments for total and p-p53 protein levels between CMV-Con and CMV-PAI-1 populations. B-C, Histograms depicting the relative expression of p53 levels (mean ± SD) for three independent studies, n = 3. D-G, Lysates of CMV-PAI-1+Con-shRNA and CMV-PAI-1+p53-shRNA double transductants are immunoblotted for p53 (D, E; P < .001), p21 (D, F; P < .01), p-H3 (D, G; P < .01), fibronectin (H, I; P < .01), collagen-1 (H, J; P < .01). Histograms in (E-G) and (I-J) depict the relative expression (mean ± SD) for indicated proteins from the immunoblots in (D) and (H), shown as biological triplicates for three independent studies (n = 3). K, Confluent monolayers of CMV-PAI-1+Con-shRNA and CMV-PAI-1+p53- shRNA HK2 cultures are serum-starved for 6 days. Phase contrast and crystal violet images are taken on day 0 and day 6 to assess cell monolayer detachment. Scale bar = 400 µm. L, Western blot analysis for FAK and p-ERK1/2 protein levels between CMV-Con and CMV-PAI-1 cultures, with ERK2 serving as a loading control, (n = 3). *P < .05, **P < .01, ***P < .001

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, shRNA, Control

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 6 Klotho downregulation consequent to PAI-1 induction contributes to epithelial dysfunction. A-C, Immunoblot analysis of CMV- Con and CMV-PAI-1 cell lysates for PAI-1 (A, B; P < .001) and klotho (A, C; P < .01) expression. Histograms in (B-C) represent the relative expression of the indicated proteins as (mean ± SD) for three independent studies (n = 3). D-I, Protein extracts of CMV-PAI-1+CMV-Con and CMV-PAI-1+CMV-Klotho double transgenic cultures are immunoblotted for klotho (D, E; P < .001), CCN2 (D, F; P < .001), p21 (D, G; P < .01), p53 (D, H; P < .001), p-SMAD3 (D, I; P < .01). GAPDH serves as loading control. Histograms in (E-I) depict the relative levels (mean ± SD) of the indicated proteins for three separate experiments (n = 3). **P < .01, ***P < .001

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Transgenic Assay, Control

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 8 Dysfunction driven by PAI-1 overexpression is independent of TGF-β1 ligand synthesis or release. A, ELISA analysis for active TGF-β1 ligand concentrations in the conditioned media isolated from serum-starved CMV-Con and CMV-PAI-1 cultures. n = 3. B-D, Cytokine protein array analysis of CMV-Con and CMV-PAI-1 conditioned media for active TGF-β1 (B), TGF-β2 (C), and TGF-β3 (D). Graphs depict the relative levels of secreted ligands (mean ± SD), n = 3. E, Immunoblot comparison of fibrotic responses in the cellular lysates of TGF-β1 stimulated or unstimulated CMV-Con and untreated CMV-PAI-1 culture extracted in parallel. F, CMV-Con HK2 cells pretreated with 20 μg/mL of TGF-β1 neutralizing antibody or 20 μg/mL IgY control antisera are stimulated with 2 ng/mL TGF-β1. Cells are harvested after 24 hours and expression of p-SMAD3, fibronectin and E-cadherin are analyzed by western blot. n = 3. G, Equally seeded CMV-PAI-1 HK2s are treated with various concentrations of TGF-β1 neutralizing antibody (0, 20, 40, 60 μg/mL) or 60 μg/mL IgY control antisera for 24 hours prior to western blot analysis of extracts for p-SMAD3, fibronectin, collagen-1, vimentin, p53 and p21, with GAPDH is serving as a loading marker. n = 3, *P < .05, **P < .01, ***P < .001, n.s., not significant. H, Western blot analysis of cell lysate extracts from CMV-PAI-1+Con shRNA and CMV-PAI-1+TGF-β1 shRNA HK2 cells for the indicated fibrotic markers. Cytokine protein array analysis of whole cell lysates (I) and conditioned media (J) used to validate TGF-β1 knockdown

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Isolation, Protein Array, Western Blot, Comparison, Control, Expressing, Marker, shRNA, Knockdown

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 9 Model. PAI-1 upregulation leads to downregulation of klotho, upregulation of p53, and induction of TGF-β1 receptor signaling independent of the TGF-β1 ligand, resulting in expression and secretion of fibrotic markers, downregulation of E-cadherin and upregulation of vimentin, leading to dedifferentiation, and upregulation of p21, p-H3, causing G2/M cell cycle arrest and a propensity to cell death, collectively establishing a role for PAI-1 in tubular epithelial dysfunction. Klotho regulates both p53 and SMAD3 signaling, promoting PAI-1-mediated tubular maladaptive responses

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: Nature

Article Title: Heterochromatin silencing of p53 target genes by a small viral protein.

doi: 10.1038/nature09307

Figure Lengend Snippet: Figure 1 | p53 is induced and phosphorylated in DE1B-55k infection but p53 activity is dominantly suppressed. a, SAECs were infected and protein lysates analysed by immunoblotting. b, U2OS cells with inducible ARF were infected as indicated and analysed for p53 levels and activation by immunoblotting. c, RT–qPCR of p53 transcriptional targets in infected SAECs (36 h.p.i.) 610 Gy c irradiation (IR). Error bars represent s.d. (n 5 3). d, Immunoblot of p53 protein phosphorylation in infected or doxorubicin (dox)-treated SAECs (36 h.p.i.). e, Immunoblot of SAECs (36 h.p.i.) infected as indicated and treated with either control (2), dox, nutlin, or TSA at 24 h.p.i.

Article Snippet: Primary antibodies were from Santa Cruz Biotechnology (p53 DO-1 and FL393, GFP, MDM2 N20), Cell Signaling Technology (phospho-p53 serine (Ser) 6, 9, 15 (16G8), 20, 33, 46, 315, 392 and threonine (Thr) 81), Upstate (p21), Calbiochem (p53 PAb1620 and 240, MDM2 2A10), Abcam (actin and histone H3), Active Motif (H3K9), Ascenion (E4-ORF3 (6A11)), ARF21, and E1B-55k (2A6).

Techniques: Infection, Activity Assay, Western Blot, Activation Assay, Quantitative RT-PCR, Irradiation, Phospho-proteomics, Control

Journal: Nature

Article Title: Heterochromatin silencing of p53 target genes by a small viral protein.

doi: 10.1038/nature09307

Figure Lengend Snippet: Figure 2 | E4-ORF3 inactivates p53 independently of E1B-55k and p53 degradation. a, SAECs were infected with the indicated viruses (detailed description in Supplementary Fig. 8) and protein lysates (36 h.p.i.) were analysed for p53 activation by immunoblotting. b, SAECs were co-infected as indicated with either a GFP control virus (Ad-GFP, 1) or a virus expressing E4-ORF3 (Ad-ORF3, 1). Protein lysates (36 h.p.i.) were analysed for p53 activation by immunoblotting. c, SAECs were infected and harvested over a 48 h time course as indicated and analysed for p53 activation by immunoblotting. d, RT–qPCR of p53 transcriptional targets in infected SAECs at 36 h.p.i. Error bars represent s.d. (n 5 3).

Article Snippet: Primary antibodies were from Santa Cruz Biotechnology (p53 DO-1 and FL393, GFP, MDM2 N20), Cell Signaling Technology (phospho-p53 serine (Ser) 6, 9, 15 (16G8), 20, 33, 46, 315, 392 and threonine (Thr) 81), Upstate (p21), Calbiochem (p53 PAb1620 and 240, MDM2 2A10), Abcam (actin and histone H3), Active Motif (H3K9), Ascenion (E4-ORF3 (6A11)), ARF21, and E1B-55k (2A6).

Techniques: Infection, Activation Assay, Western Blot, Control, Virus, Expressing, Quantitative RT-PCR

Journal: Nature

Article Title: Heterochromatin silencing of p53 target genes by a small viral protein.

doi: 10.1038/nature09307

Figure Lengend Snippet: Figure 3 | E4-ORF3 induces heterochromatin formation and prevents p53–DNA binding at endogenous promoters. a, U2OS cells were transfected with p53-luc (solid line) or p53-mutant (dashed line) luciferase plasmids and infected with indicated viruses. Luminescence is plotted against time. b, c, U2OS cells were infected as indicated or treated with doxorubicin. b, p53 induction was analysed by immunoblotting and p53 transcriptional targets quantified by RT–qPCR (36 h.p.i.). Error bars represent s.d. (n 5 3) c, p53 ChIPs were analysed by semiquantitative PCR for p21 and MDM2 promoter sequences. d, p53 (green) and H3K9me3 (red) immunofluorescence of infected U2OS cells (36 h.p.i.). e, Localization of SUV39H1, SUV39H2, SETDB1 and G9a (green) with H3K9me3 (red) in D55k-infected U2OS cells (36 h.p.i.).

Article Snippet: Primary antibodies were from Santa Cruz Biotechnology (p53 DO-1 and FL393, GFP, MDM2 N20), Cell Signaling Technology (phospho-p53 serine (Ser) 6, 9, 15 (16G8), 20, 33, 46, 315, 392 and threonine (Thr) 81), Upstate (p21), Calbiochem (p53 PAb1620 and 240, MDM2 2A10), Abcam (actin and histone H3), Active Motif (H3K9), Ascenion (E4-ORF3 (6A11)), ARF21, and E1B-55k (2A6).

Techniques: Binding Assay, Transfection, Mutagenesis, Luciferase, Infection, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: Nature

Article Title: Heterochromatin silencing of p53 target genes by a small viral protein.

doi: 10.1038/nature09307

Figure Lengend Snippet: Figure 5 | p53 transcriptional targets are silenced selectively in the backdrop of global transcriptional changes that drive oncogenic cellular and viral replication. Affymetrix global gene expression analyses of SAECs. a, Heat map of the 1,730 overlapping differentially regulated genes (log-fold change .2 or ,22 with a false discovery rate (FDR) of 0.05) between D55k/ DORF3- and D55k- versus mock-infected SAECs (36 h.p.i.). b, Unsupervised hierarchical clustering of 46 top differentially upregulated transcripts in both D55k/DORF3 infection and nutlin treatment. c, Pie-chart depicting the percentage of upregulated transcripts (log-fold change .2 and FDR of 0.05) in D55k/DORF3 versus D55k that have predicted p53 transcription factor binding sites and/or induced by a log-fold change .1.5 in response to nutlin. d, Summary and model.

Article Snippet: Primary antibodies were from Santa Cruz Biotechnology (p53 DO-1 and FL393, GFP, MDM2 N20), Cell Signaling Technology (phospho-p53 serine (Ser) 6, 9, 15 (16G8), 20, 33, 46, 315, 392 and threonine (Thr) 81), Upstate (p21), Calbiochem (p53 PAb1620 and 240, MDM2 2A10), Abcam (actin and histone H3), Active Motif (H3K9), Ascenion (E4-ORF3 (6A11)), ARF21, and E1B-55k (2A6).

Techniques: Gene Expression, Infection, Binding Assay