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: Reproduction

Article Title: Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes

doi: 10.1530/rep-17-0687

Figure Lengend Snippet: Figure 2. Western blot analysis of p53 and p-p53 in fallopian tubes (FT) Ctr: Control mice; 6R and 8R: mice undergoing 6 rounds and 8 rounds of gonadotropin stimulation (A). Data

Article Snippet: Mouse monoclonal 89 p53 and OCT3/4, rabbit polyclonal Cyclin D1, phospho-B-catenin (Thr41/Ser45), B-catenin, 90 5 phospho-AKT (Ser473), AKT, phospho-GSK3B (Ser9), GSK3B and SOX2 primary 91 antibodies were purchased from Santa Cruz Biotechnology (CA, USA); mouse monoclonal 92 phospho-p53 (Ser15), rabbit monoclonal phospho-CHK1 (Ser345) and phospho-H2A.X 93 (Ser139) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Control

Journal: PloS one

Article Title: Cucurbitacin B induced ATM-mediated DNA damage causes G2/M cell cycle arrest in a ROS-dependent manner.

doi: 10.1371/journal.pone.0088140

Figure Lengend Snippet: Figure 4. ATM knocked down reversed Cuc B induced G2/M phase arrest. A549 cells transfected with 100 pmol ATM siRNA or negative control siRNA for 12 h followed by treatment with or without 200 nM Cuc B for another 24 h. The cell cycle was analyzed (A, B). A549 cells transfected with ATM siRNA and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Chk1 on Ser-345, Cdc25C on Ser-216, p53 on Ser-15 and protein levels of p53 Cyclin B1 were analyzed by Western blot (C). Total Cyclin B1 protein was immunoprecipitated from ATM siRNA transfected and treated with or without 200 nM Cuc B A549 cells lysates and analyzed for Cdk1 for 24 h (D). *p,0.05 vs. Cont. Cont, control group. doi:10.1371/journal.pone.0088140.g004

Article Snippet: Specific antibodies PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e88140 against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phosphop53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology (USA).

Techniques: Transfection, Negative Control, Phospho-proteomics, Western Blot, Immunoprecipitation, Control

Journal: PloS one

Article Title: Cucurbitacin B induced ATM-mediated DNA damage causes G2/M cell cycle arrest in a ROS-dependent manner.

doi: 10.1371/journal.pone.0088140

Figure Lengend Snippet: Figure 7. Schematic representation of the signaling pathways involved in the Cuc B induced G2/M checkpoint in A549 cells. Cuc B induced ROS formation, which lead to DNA damage. In response to DNA damage, ATM via autophosphorylation activated G2/M checkpoint. The downstream effectors of ATM, Chk1 and p53, were both activated, which resulted in Cdc25C phosphorylation and increased expression of 14-3-3-s respectively. Phosphorylation of Cdc25C on Ser-216 by activated Chk1 facilitated the binding of 14-3- 3-s to Cdc25C, which prevents Cdk1 dephosphorylation, and keeps Cdk1-Cyclin B1 complex inactivated, causing G2/M phase arrest. doi:10.1371/journal.pone.0088140.g007

Article Snippet: Specific antibodies PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e88140 against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phosphop53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology (USA).

Techniques: Protein-Protein interactions, Phospho-proteomics, Expressing, Binding Assay, De-Phosphorylation Assay

Journal: Nature Communications

Article Title: Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties

doi: 10.1038/s41467-020-16050-w

Figure Lengend Snippet: a Genes differentially expressed in IMR90 cells upon treatment with cNSF-HA or fNSF-HA (HA polymer length-dependent genes). Cells were pre-incubated for 6 h with 20 μg/ml cNSF-HA, fNSF-HA, or PBS and then HA was removed and cells were exposed to 200 μM tBHP for 1 h. Cells were collected 6 h after starting the tBHP-treatment. b Enrichment of GO Term, Reactome pathway, and transcription factor protein-protein interactions (PPIs) in the HA polymer length-dependent genes. c , d Enrichment of transcription factor binding sites (TFBS) in the HA polymer length-dependent genes. White bars represents the enrichment of TFBS in all HA polymer length-dependent genes. Gray bars represents the enrichment of TFBS in HA polymer length-dependent genes that are involved in the transcriptional regulation by p53 or encoding p53-interacting proteins. Black bars represents the enrichment of TFBS in HA polymer length-dependent p53 target genes. e Differential expression levels of the HA polymer length-dependent genes (defined in the presence of tBHP) in IMR90 cells incubated for 6 h with 20 μg/ml cNSF-HA, fNSF-HA, or PBS. f The violin plot represents the distribution of p -values of a two-tailed t -test comparing the gene expression levels of the HA polymer length-dependent genes and HA-polymer length-independent genes (defined in the presence of tBHP) between cNSF-HA- and fNSF-HA-incubated IMR90 cells. Cells were incubated for 6 h with 20 μg/ml cNSF-HA or fNSF-HA without further tBHP-treatment. g Differential expression levels of the HA polymer length-dependent genes (defined in the presence of tBHP) in control and CD44-overexpressing IMR90 cells incubated for 6 h with 20 μg/ml cNSF-HA or PBS.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked with 5% BSA or 5% milk in TBST for 1 h, and probed with the following primary antibodies: anti-β-actin (sc-47778; Santa Cruz), anti-CD44 (ab157107; abcam), anti-FLAG (M2; sigma), anti-GFP (for YFP detection) (ab6556; abcam), anti-H3 (#9715; CST), anti-p53 (human) (10442-1-AP; Proteintech), anti-p53 (mouse) (ab26; Abcam), anti-phospho-p53 (Ser-9) (#9288; CST), anti-phospho-p53 (Ser-15) (#9286; CST), anti-phospho-p53 (Ser-46) (#2521; CST), and anti-phospho-p53 (Thr-81) (#2676; CST).

Techniques: Polymer, Incubation, Protein-Protein interactions, Binding Assay, Quantitative Proteomics, Two Tailed Test, Gene Expression, Control

Journal: Nature Communications

Article Title: Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties

doi: 10.1038/s41467-020-16050-w

Figure Lengend Snippet: a Western blots showing p-p53-Ser9 levels in IMR90 cells treated with 20 μg/ml cNSF-HA or fNSF-HA for 6 h. b Quantification of Western blots shown in ( a ) by Image J. Averages are of three biological replicates. For each biological replicate, immunoblotting was performed three times and averaged. c p53-knockdown abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced cell death in IMR90 cells. The percentages of live cells were measured by Annexin-V/PI staining 1 day after 1 h of 3 mM tBHP-treatment ( n = 3). Cells were pre-incubated for 6 h with 20 μg/ml NSF-HA or PBS before tBHP-treatment. siRNA was transfected 2 days before the first tBHP-treatment. d p53-knockdown abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced growth arrest in IMR90 cells ( n = 4). Cells were pre-incubated for 6 h with 20 μg/ml NSF-HA or PBS for 6 h and then HA was removed and cells were exposed to 200 μM tBHP for 1 h on day 0 and day 1. siRNA was transfected 2 days before day 0. e Confirmation of p53-knockdown in IMR90 cells by Western blot. Cells were collected 2 days after p53 or control siRNA transfection. Immunoblotting was repeated once with similar result. f p53-knockout abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced cell death in MSF. The percentages of live MSF were measured by Annexin-V/PI staining 1 day after 1 h of 1.5 mM tBHP-treatment ( n = 3). Cells were pre-incubated for 6 h with 20 μg/ml NSF-HA or PBS before tBHP-treatment. g p53-knockout abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced growth arrest in MSF ( n = 4). Cells were pre-incubated for 6 h with 20-μg/ml NSF-HA or PBS for 6 h and then HA was removed and cells were exposed to 150-μM tBHP for 1 h on day 0 and day 1. h Confirmation of p53-knockout in MSF by Western blot. Immunoblotting was repeated once with similar result. Error bars are presented as mean ± SD values. * p < 0.05 (two-tailed t -test).

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked with 5% BSA or 5% milk in TBST for 1 h, and probed with the following primary antibodies: anti-β-actin (sc-47778; Santa Cruz), anti-CD44 (ab157107; abcam), anti-FLAG (M2; sigma), anti-GFP (for YFP detection) (ab6556; abcam), anti-H3 (#9715; CST), anti-p53 (human) (10442-1-AP; Proteintech), anti-p53 (mouse) (ab26; Abcam), anti-phospho-p53 (Ser-9) (#9288; CST), anti-phospho-p53 (Ser-15) (#9286; CST), anti-phospho-p53 (Ser-46) (#2521; CST), and anti-phospho-p53 (Thr-81) (#2676; CST).

Techniques: Western Blot, Knockdown, Staining, Incubation, Transfection, Control, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties

doi: 10.1038/s41467-020-16050-w

Figure Lengend Snippet: Our data indicate that vHMM-HA attenuates CD44 protein-protein interactions (PPI), whereas HMM-HA promotes them. Concomitantly, vHMM-HA suppresses CD44-dependent gene expression, including those regulated by CD44-dependent transcription factors such as ELK, EGR1, and CD44-ICD. vHMM-HA and HMM-HA thereby induce opposing effects on the expression of CD44-dependent genes, which are also associated with the p53 pathway. As a result, vHMM-HA partially attenuates p53 and its target genes, and protects cells in a p53-dependent manner.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked with 5% BSA or 5% milk in TBST for 1 h, and probed with the following primary antibodies: anti-β-actin (sc-47778; Santa Cruz), anti-CD44 (ab157107; abcam), anti-FLAG (M2; sigma), anti-GFP (for YFP detection) (ab6556; abcam), anti-H3 (#9715; CST), anti-p53 (human) (10442-1-AP; Proteintech), anti-p53 (mouse) (ab26; Abcam), anti-phospho-p53 (Ser-9) (#9288; CST), anti-phospho-p53 (Ser-15) (#9286; CST), anti-phospho-p53 (Ser-46) (#2521; CST), and anti-phospho-p53 (Thr-81) (#2676; CST).

Techniques: Protein-Protein interactions, Gene Expression, Expressing

Journal: Cells

Article Title: Towards Understanding the Development of Breast Cancer: The Role of RhoJ in the Obesity Microenvironment

doi: 10.3390/cells13020174

Figure Lengend Snippet: Different antibodies used and their dilutions.

Article Snippet: Phospho-p53 (S15) , 1:1000 , Rabbit , Cell signaling, Danvers, MA, USA , 9286s.

Techniques:

Journal: Cancer Research

Article Title: Homeodomain-Interacting Protein Kinase 2 Is the Ionizing Radiation–Activated p53 Serine 46 Kinase and Is Regulated by ATM

doi: 10.1158/0008-5472.can-06-2884

Figure Lengend Snippet: Figure 1. HIPK2 accumulates after ionizing radiation and forms a complex with p53. HepG2 (A) and HCT116 (B) cells were irradiated with 12 Gy and harvested at the given time points posttreatment, and total cell extracts were analyzed by immunoblotting. C, HCT116 p53+/+ and HCT116 p53/ cells were irradiated as indicated, and total cell extracts were analyzed by immunoblotting. D, reverse transcription-PCR analysis of HIPK2 and h-actin expression in untreated and irradiated HepG2 cells. Cells were harvested 24 h posttreatment, and total RNA was used for the reverse transcription-PCR reaction. E, total cell lysates from HepG2 cells either left untreated or irradiated with 16 Gy were subjected to immunoprecipitation using rabbit IgG control antibodies or affinity-purified rabbit HIPK2 antibodies. The precipitated complexes were analyzed by immunoblotting with HIPK2 and p53 specific antibodies. Signals were quantified using the ImageQuant software package. Representative of three (A–D) or two (E) independent experiments.

Article Snippet: The following antibodies were used: p53 (DO-1) and glyceraldehyde3-phosphate dehydrogenase (Santa Cruz, Inc., Heidelberg, Germany), a-tubulin (Sigma, Munich, Germany), p53 phosphorylated Ser46 and phosphorylated Ser15 (Cell Signaling Technologies, Danvers, MA), p53DINP1 (Novus Biologicals, Littleton, CO), actin clone C4 (MP Biomedicals, Illkirch, France).

Techniques: Irradiation, Western Blot, Reverse Transcription, Expressing, Immunoprecipitation, Control, Affinity Purification, Software

Journal: Cancer Research

Article Title: Homeodomain-Interacting Protein Kinase 2 Is the Ionizing Radiation–Activated p53 Serine 46 Kinase and Is Regulated by ATM

doi: 10.1158/0008-5472.can-06-2884

Figure Lengend Snippet: Figure 2. IR-induced HIPK2 accumulation and activation correlates with p53 Ser46

Article Snippet: The following antibodies were used: p53 (DO-1) and glyceraldehyde3-phosphate dehydrogenase (Santa Cruz, Inc., Heidelberg, Germany), a-tubulin (Sigma, Munich, Germany), p53 phosphorylated Ser46 and phosphorylated Ser15 (Cell Signaling Technologies, Danvers, MA), p53DINP1 (Novus Biologicals, Littleton, CO), actin clone C4 (MP Biomedicals, Illkirch, France).

Techniques: Activation Assay

Journal: Cancer Research

Article Title: Homeodomain-Interacting Protein Kinase 2 Is the Ionizing Radiation–Activated p53 Serine 46 Kinase and Is Regulated by ATM

doi: 10.1158/0008-5472.can-06-2884

Figure Lengend Snippet: Figure 3. HIPK2 down-regulation specifically inhibits IR-induced p53 Ser46

Article Snippet: The following antibodies were used: p53 (DO-1) and glyceraldehyde3-phosphate dehydrogenase (Santa Cruz, Inc., Heidelberg, Germany), a-tubulin (Sigma, Munich, Germany), p53 phosphorylated Ser46 and phosphorylated Ser15 (Cell Signaling Technologies, Danvers, MA), p53DINP1 (Novus Biologicals, Littleton, CO), actin clone C4 (MP Biomedicals, Illkirch, France).

Techniques:

Journal: Scientific Reports

Article Title: Regulation of cardiomyocyte DNA damage and cell death by the type 2A protein phosphatase regulatory protein alpha4

doi: 10.1038/s41598-021-85616-5

Figure Lengend Snippet: Regulation of the DNA damage response by α4 protein expression in cardiomyocytes. H9c2 cardiomyocytes were transfected with either 50 nM non-targeting control (NTC) siRNA or 50 nM siRNA specific to rat α4 protein (siα4) for 1–4 days (n = 4) and the KD of α4 protein expression was confirmed by Western analysis using an antibody raised against human α4 ( a ). α4 protein expression was knocked down for 4 days and Western analysis (short and long exposures) was used to probe samples (n = 3) with an antibody specific to phosphorylated S / T QG motifs within ATM/ATR substrates. Arrows indicate substrates whose phosphorylation status (n = 3) was elevated in response to α4 KD ( b ). The expression and phosphorylation (Ser15) of p53 were determined by Western analysis using rodent-specific antibodies raised against mouse p53 protein in samples following 4 days of α4 KD, n = 5 ( c ). The expression of PARP was determined by Western analysis (short and long exposures) following 1–4 days of α4 KD, n = 3. Arrows indicate full length PARP (116 kDa) and cleaved fragments (89 kDa and 24 kDa) ( d ). All data represents data mean values ± SEM and statistical significance vs. non-targeting control (NTC) was determined by a one-tailed Student’s T-test. Expression of β-actin was used to confirm equal protein loading between samples.

Article Snippet: Antibodies to detect Bax (#2772), Bcl-xL (#2762), phosphospecific ATM/ATR substrate (#6966), IGBP1 (α4) (#5699), PARP (#9542), phosphospecific (Ser139) H2AX (#9718), H2AX (#2595), rodent-specific p53 (#32532), rodent-specific phosphospecific (Ser15) p53 (#12571) and V5-tag (#13202) were obtained from Cell Signaling Technology (USA).

Techniques: Expressing, Transfection, Control, Western Blot, Phospho-proteomics, One-tailed Test