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: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Growth curves of Cas9-expressing HCT116 in the genome wide CRISPR/Cas9 knockout screen shown in . At each plotted point, cells were treated for 24 hours with vehicle or 1 µM MM17 and allowed to recover without treatment until 80% confluency (n=1). B. Immunoblots of p53 and actin (loading control) in HCT116 mCherry mock, ZsGreen p53 KO guide 1 (p53 KO-1), and ZsGreen p53 KO guide 2 (p53 KO-2) cells. C. Immunoblots of p21 and tubulin (loading control) in HCT116 mCherry mock, and ZsGreen p21 KO cells. D, E. Growth competition assay between mCherry mock and ZsGreen mock, ZsGreen p53 KO-1, ZsGreen p53 KO-2, or ZsGreen p21 KO cells. At each plotted point, cells were treated for 24 hours with MM17 ( D ) or paclitaxel ( E ) and allowed to recover without compound until confluency. (n=3 biological replicates, mean ± sem). F. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem). G. Immunoblot of MDM2 and Ponceau S staining (loading control) in HCT116 p53 KO cells transduced with non-target control or four sgRNAs targeting MDM2. sgMDM2 guide 3 was selected for subsequent experiments. H. Cresyl violet staining after 1 week of antibiotics selection of HCT116 cells expressing empty vector, ectopic MDM2 WT, or MDM2 C311F transduced with mock or MDM2 guide. I. Immunoblots of MDM2 and actin (loading control) of cell lines in (H). J. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem). K. Viability assay of HCT116 cells treated with etoposide for 72 hours. (n = 3 biological replicates, mean ± sem). L. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Expressing, Genome Wide, CRISPR, Knock-Out, Western Blot, Control, Competitive Binding Assay, Quantitation Assay, Staining, Transduction, Selection, Plasmid Preparation, Viability Assay

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Genome wide CRISPR/Cas9 knockout screen in HCT116 cells. The log2 fold change for each gene comparing samples treated with vehicle or 1 µM MM17 was determined by averaging the log2 fold changes of its associated sgRNAs and subtracting the average log2 fold change of non-targeting sgRNAs. The top two most enriched genes, and one of the biogenesis factors removed by Rix7 are indicated in red and labeled. B. Immunoblots of p53, p21, and tubulin (loading control) in HCT116 NVL WT and NVL R403W CRISPR cells treated with 3 µM MM17. C . Cryo-EM structure of the MDM2 299-340 –5S RNP complex (PDB ID - 8BGU; MDM2 - orange, uL5 - turquoise, uL18 - red, 5S rRNA - pink) . MDM2 C311 residue (yellow) and Zn 2+ (purple) at the MDM2-uL5 binding interface are labeled. D, E. Immunoblots of p53, p21 and actin (loading control) in HCT116 MDM2 WT and MDM2 C311F cells treated with MM17 ( D ) or etoposide (E) for 24 hours. F. Cell cycle analysis of HCT116 mock and p53 KO cells treated with 3 µM MM17. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, ns = non-significant). G. Live cell imaging analysis of relative cell area of HCT116 mock and p53 KO cells treated with 3 µM MM17 for 72 hours. (n = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Genome Wide, CRISPR, Knock-Out, Labeling, Western Blot, Control, Cryo-EM Sample Prep, Residue, Binding Assay, Cell Cycle Assay, Two Tailed Test, Live Cell Imaging

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Immunoblots and quantitation of caspase-3 and actin (loading control) in HCT116 cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). B. Flow cytometry-based cell cycle analysis in HCT116 cells treated with vehicle or 3 µM MM17 for 3 days. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, *p<0.05). C. Immunoblots of p53 and actin (loading control) in HCT116 mock engineered or p53 knockout (KO) cells. D. Viability (log 2 fold change relative to vehicle) of 855 cancer cell lines treated with 10 µM MM17, grouped by their p53 mutation or copy number deletion status. Each dot represents a cell line. (two-tailed paired t-test, ****p<0.0001).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Quantitation Assay, Control, Flow Cytometry, Cell Cycle Assay, Two Tailed Test, Knock-Out, Mutagenesis

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A bioavailable analog MM927 inhibits colorectal and leukemia tumor xenograft growth in vivo without overt toxicity. A. Chemical structure of MM927 and the potency (IC50) of its impact on the viability of HCT116. B. Pharmacokinetic analysis of MM927 in mouse plasma after a 10 mg/kg intraperitoneal (IP) injection (n = 3 mice/group, mean ± sem). C, D. Representative microscopy images ( C ) and quantitation ( D ) showing immunofluorescence of p53 (magenta), Ki-67 (green), and nucleus (DAPI, blue) in NVL WT and NVL R403W CRISPR tumor xenografts implanted in either flank of the same mouse. Mice were treated with 35 mg/kg MM927 IP twice daily for 3 days, and tumors were harvested 6 hours after the last dose. (Scale bar = 10 µm; n = 5 mice/group, mean ± sem). E. Effect of MM927 (35 mg/kg IP twice daily for 21 days) on HCT116 NVL WT or NVL R403W CRISPR tumor xenograft growth (n = 10 mice/group, mean ± sem). F . Tumor volume measurements in (E) at the end of 21 days or euthanasia due to tumor diameter exceeding 2 cm (n = 10 mice per group, mean ± sem; two-tailed unpaired t-test, ** p < 0.01, ns = non-significant). G. Body weight % changes in HCT116-tumor bearing mice at the end of MM927 treatment (n = 20 mice/group, mean ± sem; two-tailed unpaired t-test, ns = non-significant). H. Levels of hemoglobin, white blood cells, platelets, liver enzymes AST and ALT, and renal clearance of blood urea nitrogen (BUN) 3 hours after the last dose (n = 10 mice per group, mean ± sem; two-tailed unpaired t-test, **p<0.01, *p<0.01, ns = non-significant). I. Representative images and quantitation of leukemic disease burden by bioluminescent imaging of luciferase expressing MOLM-13 cells. (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, *** p < 0.001). J, K . Flow cytometry-based quantitation of MOLM-13 cells (human CD45 positive) in the mouse bone marrow ( J ) and peripheral blood ( K ) (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, ****p<0.0001). L . Body weight changes at the end of MM927 treatment in mice xenografted with MOLM-13 (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, ns = non-significant).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: In Vivo, Clinical Proteomics, Injection, Microscopy, Quantitation Assay, Immunofluorescence, CRISPR, Two Tailed Test, Imaging, Luciferase, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Polysome profiling of HCT116 NVL WT ( A ) and NVL R403W CRISPR ( B ) cells treated with 0.5 µM MM927 for 24 hours. B, C. Representative microscopy images and quantitation of existing (green) and newly synthesized (magenta) eL36-SNAP in NVL WT ( B ) and NVL R403W CRISPR ( C ) cells treated with 0.5 µM MM927 for 24 hours. (Scale bar = 10 µm; n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, **p<0.01, ns = non-significant). Data from the vehicle group is re-used from these are done in the same experiment. D. Representative microscopy images and quantitation of existing (green) and newly synthesized (magenta) eS17-SNAP in HCT116 cells treated with 0.5 µM MM927 for 24 hours. (Scale bar = 10 µm; n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, **p<0.01, ns = non-significant). Data from the vehicle group is re-used from , as these are done in the same experiment. E. Flow cytometry-based analysis of cell cycle profile in HCT116 mock and p53 KO cells treated with 0.5 µM MM927. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, *p<0.05, ns = non-significant). F. Viability assay of HCT116 NVL R403W CRISPR cells treated with MM927 for 72 hours. (N = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: CRISPR, Microscopy, Quantitation Assay, Synthesized, Two Tailed Test, Flow Cytometry, Viability Assay

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: MM927 studies in HCT116 tumor xenografts. A. Immunoblots of Flag (NVL) and tubulin (loading control) in HCT116 NVL AID/AID cells ectopically expressing empty vector, human NVL, or mouse NVL treated with Ph-IAA for 6 hours. B. Viability assay of cell lines in (A) treated with 0.03 µM Ph-IAA for 72 hours (n = 2 biological replicates, mean ± sem). C. Viability assay of HCT116 NVL AID/AID cells ectopically expressing human NVL or mouse NVL in treated with MM927 and 0.03 µM Ph-IAA for 72 hours (n = 2 biological replicates; mean ± sem). D, E. Immunoblots ( D ) and quantitation ( E ) of p53, p21, and tubulin (loading control) of HCT116 NVL WT and NVL R403W CRISPR tumor xenografts implanted in either flank of the same mouse treated with one 35 mg/kg MM927 IP injection. Right two lanes are in vitro samples of HCT116 cells treated with 10 µM MM17 for 24 hours. (n=2 to 3 biological replicates, mean ± sem). F. Individual tumor volume measurements in . Each line represents a mouse. G. Pharmacokinetic quantitation of MM927 3 hours after the last dose in the mouse plasma and tumors (n = 10 mice/group, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Control, Expressing, Plasmid Preparation, Viability Assay, Quantitation Assay, CRISPR, Injection, In Vitro, Clinical Proteomics

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Immunoblots of p53 and actin (loading control) in MOLM-13 mock engineered or p53 knockout (KO) cells. B. Viability assay of cell lines in (A) treated with MM17 for 72 hours. (n = 3 biological replicates, mean ± sem). C. Immunoblots and quantitation of caspase-3 and actin (loading control) in MOLM-13 mock and p53 KO cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). D. Flow cytometry-based apoptosis analysis and quantitation in MOLM-13 mock and p53 KO cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). E. Schematic illustrating the proposed mechanism of cellular response to MM17. F. Immunoblots of NVL and actin (loading control) in MOLM-13 cells expressing empty vector, NVL WT, or NVL R403W. G. Growth curves of cell lines in (F). n = 3 biological replicates, mean ± sem). H. Viability assay of cell lines in (F) treated with MM927 for 72 hours. (n = 2 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Control, Knock-Out, Viability Assay, Quantitation Assay, Flow Cytometry, Expressing, Plasmid Preparation

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: Profiling the Oxylipidome in Aged Mice after Chronic Ethanol Feeding: Identifying Lipid Metabolites as Drivers of Hepatocyte Stress

doi: 10.1016/j.alcohol.2022.08.012

Figure Lengend Snippet: Young and aged adult WT mice were allowed free access to ethanol (32%,d25) or pair-fed control diets. A) Liver RNA was isolated and expression of senescence markers, p16 and p21 mRNA was detected in mouse livers using qRT-PCR. B/C) Paraffin-embedded liver sections were deparaffinized followed by immunodetection of PCNA and p53 and nuclei were counterstained with hematoxylin. Frozen liver sections were stained for β-galactosidase activity. Images were acquired using a ×10 objective and positive staining was quantified using Image-J. Data are from two independent trials; individual values are reported and represented as means ± SEM, n=8 pair-fed and 12 EtOH-fed mice/group. *Values were significantly different from each other (P<0.05).

Article Snippet: Paraffin-embedded liver sections were deparaffinized and stained with antibodies against NIMP-R14 (Novus Biologicals, cat NB600–1387), PCNA (Millipore, cat# MAB424R) and p53 (Novus Biologicals, cat# NB200–103).

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Immunodetection, Staining, Activity Assay

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Targeting strategy. The wildtype (WT) Trp53 gene is within a 17-kb-long EcoRI (RI) fragment (black boxes are for coding sequences and white boxes for UTRs). The targeting construct contains: (1) a 1.5-kb-long 5’ homology region; (2) a Lox-Stop-Lox (LSL) cassette with a neomycin selection gene (Neo), four transcriptional stops (STOP) and an EcoRI site, flanked by LoxP sites (arrowheads); (3) p53 coding exons, including the Y217C (YC) missense mutation in exon 6 (asterisk) and an additional BanII site; (4) a 2.8-kb-long 3’ homology region; and (5) the diphteria α-toxin (DTA) gene for targeting enrichment. Proper recombinants with a Trp53 LSL-Y217C allele, resulting from the described crossing-overs, were G418 resistant. They were identified by a 2.4-kb-long band after PCR with primers a and b, and confirmed by bands of 635 and 224 bp after PCR with primers c and d and BanII digestion. They were also verified by Southern blot with the indicated probe as containing a 10.5 kb EcoRI band. Two recombinant ES clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping with primers c and d and BanII digestion. Excision of the LSL cassette was performed in vivo, by breeding Trp53 +/LSL-Y217C male mice with females carrying the PGK- Cre transgene, to obtain mice with a Trp53 Y217C allele. ( B–D ) Screening of recombinant ES clones (+) by PCR with primers a and b ( B ); PCR with primers c and d then BanII digestion ( C ); Southern blot ( D ). ( E ) Genotyping of mouse embryonic fibroblasts (MEFs) from an intercross of Trp53 +/Y217C mice, by PCR with primers c and d and BanII digestion. ( F ) Trp53 Y217C sequence around codon 217. The introduced Y217C missense mutation and the silent mutation creating an additional BanII restriction site are highlighted (asterisks). ( G ) WT and Trp53 Y217C/Y217C (YC/YC) MEFs express similar p53 mRNA levels. Total RNA was extracted, then p53 mRNAs were quantified by real-time qPCR, normalized to control mRNAs and the amount in WT cells was assigned the value of 1. Means + SEM (n=3) are shown. Primer sequences are listed in . Figure 1—source data 1. Labeled files for gels and blots in . Figure 1—source data 2. Raw and unedited gels and blots for .

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Construct, Selection, Mutagenesis, Southern Blot, Recombinant, Clone Assay, Injection, Transmission Assay, In Vivo, Sequencing, Control, Labeling

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: Portions of the DNA-binding domains from the mouse (residues 208–228) and human (residues 211–231) p53 proteins are shown, with identical residues in bold, and mouse Tyrosine 217 and human Tyrosine 220 in red.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Binding Assay

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Increased p53 protein levels in Trp53 YC/YC and Trp53 +/YC mouse embryonic fibroblasts (MEFs). MEFs of the indicated genotypes were treated or not with 10 μM Nutlin 3a for 24 hr, then protein extracts were immunoblotted with antibodies against Mdm2, p53, p21, and actin. ( B ) The transactivation of classical p53 target genes Cdkn1a and Mdm2 is impaired in Trp53 YC/YC cells. Wildtype (WT), Trp53 YC/YC , and Trp53 -/- MEFs were treated as in ( A ), then (top) mRNAs were quantified in five to six independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed WT cells assigned a value of 1; or (bottom) ChIP assays were performed at the Cdkn1a and Mdm2 promoters in two to three independent experiments with an antibody against p53 or rabbit IgG as a negative control. Immunoprecipitates were quantified using real-time PCR, normalized to data over an irrelevant region, and the amount in unstressed WT cells was assigned a value of 1. Error bars: SEM. ( C ) Assessment of p53 WT and p53 Y217C subcellular localization by cellular fractionation. WT and Trp53 YC/YC MEFs were treated or not with 1 μΜ doxorubicin (Doxo) for 24 hr, submitted to cellular fractionation, then protein extracts were immunoblotted with antibodies against p53 or the fraction controls Tubulin for cytoplasm (Cp.), Nup98 for nucleoplasm (Np.), and histone H3 for chromatin (χin). ( D ) Assessment of p53 WT and p53 Y217C subcellular localization by immunofluorescence. WT, Trp53 YC/YC and Trp53 -/- MEFs were treated with 10 μM Nutlin 3a for 24 hr, then stained with antibodies against p53 (red) or actin (green) and DNA was counterstained with DAPI (blue). ( E ) Absence of a cell cycle arrest response in Trp53 YC/YC MEFs. Asynchronous cell populations of Trp53 +/+ , Trp53 YC/YC , and Trp53 -/- MEFs were analyzed 24 hr after 0, 3, or 12 Gy γ-irradiation. Means + SEM from three independent experiments. ( F ) Absence of a p53-dependent apoptotic response in Trp53 YC/YC thymocytes. Age-matched mice of the indicated genotypes were left untreated or submitted to 10 Gy whole-body γ-irradiation then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS. Means + SEM from two independent experiments. ( G ) Increased chromosomal instability in Trp53 YC/YC fibroblasts. Metaphase spreads were prepared from WT, Trp53 YC/YC , and Trp53 -/- MEFs at passage 4, then aberrant metaphases (with chromosome breaks, radial chromosomes, or double-minute chromosome [DMs]) were scored. Left: distribution of aberrant metaphases. Data from 110 WT, 97 Trp53 YC/YC , or 119 Trp53 -/- complete diploid metaphases, independently observed by two experimenters. Right: examples of two aberrant Trp53 YC/YC metaphases: one with a DM, a chromosome break (Br) and a radial chromosome (R), the other with multiple DMs. Enlargements of regions of interest are presented between the two metaphases. Scale bars ( D, G ): 5 μm. ***p<0.001, **p<0.01, *p<0.05, °p=0.09, ns: non-significant by Student’s t ( B, E, F ) or Fisher’s ( G ) tests. Figure 2—source data 1. Labeled files for gels and blots in . Figure 2—source data 2. Raw and unedited gels and blots for .

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Real-time Polymerase Chain Reaction, Control, Negative Control, Cell Fractionation, Immunofluorescence, Staining, Irradiation, Labeling

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Transactivation of the classical p53 target genes Cdkn1a (alias p21 ), Mdm2, and Pmaip1 (alias Noxa ) in response to Nutlin or Doxorubicin. WT, Trp53 +/YC , and Trp53 +/- mouse embryonic fibroblasts (MEFs) were treated or not with 10 μM Nutlin 3a (Nut) or 1 μM Doxorubicin (Doxo) for 24 hr, then mRNAs were quantified in ≥4 independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed WT cells assigned a value of 1. Means + SEM are shown. For each condition and gene, a dominant-negative effect (DNE) would lead to significant decrease in transactivation in Trp53 +/YC MEFs compared to both WT and Trp53 +/- MEFs, a result that was not observed. ( B ) Cell cycle arrest responses to γ-irradiation. Asynchronous cell populations of WT, Trp53 +/YC , and Trp53 +/- MEFs were analyzed 24 hr after 0, 3, or 12 Gy γ-irradiation. Means + SEM from three independent experiments. The comparison of cells submitted to identical irradiation doses revealed similar arrest responses in cells of all genotypes. ( C ) Apoptotic responses to γ-irradiation. WT, Trp53 +/YC , and Trp53 +/- MEFs age-matched mice were left untreated or submitted to 10 Gy whole-body γ-irradiation, then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS. Means + SEM from two independent experiments. The percentage of apoptotic cells was significantly higher in irradiated WT thymocytes compared to irradiated Trp53 +/YC or Trp53 +/- cells, whereas Trp53 +/YC and Trp53 +/- cells were not significantly different. **p<0.01, *p<0.05, ns: non-significant by Student’s t-test.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Real-time Polymerase Chain Reaction, Control, Dominant Negative Mutation, Irradiation, Comparison, Staining

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Distribution of weaned mice obtained from Trp53 +/- or Trp53 +/YC intercrosses. Obs: observed numbers of mice at weaning (P21); exp: expected numbers assuming a Mendelian distribution without sex distortion; f/m: observed female/male ratios. Consistent with previous reports, the observed distribution of weaned mice from Trp53 +/- intercrosses did not conform to values expected for a Mendelian distribution without sex distortion (U=5; χ 2 =16.31>15.09), indicating a significant deficit in female Trp53 -/- mice (top). The distribution of weaned mice from Trp53 +/YC intercrosses diverged even more from values for a Mendelian distribution without sex distortion (U=5; χ 2 =104.23>15.09), due to a striking deficit in female Trp53 YC/YC mice (bottom). Differences between the frequencies of Trp53 YC/YC (4/677) and Trp53 -/- (8/196) females in the progeny, or between the female to male ratios for Trp53 YC/YC (4/75) and Trp53 -/- (8/28) animals, are statistically significant (p=0.0012 and p=0.0087 in Fisher’s tests, respectively). ( B ) Exencephaly is frequently observed in p53 YC/YC female embryos. Top: the distribution of E12.5–16.5 embryos from heterozygous ( Trp53 +/YC ) intercrosses or heterozygous-homozygous ( Trp53 +/YC × Trp53 YC/YC ) crosses is shown. f or m exenc.: number of female or male embryos with exencephaly; o.a.: embryos with other abnormalities. Below, examples of female Trp53 YC/YC embryos at E12.5, E13.5, and E16.5 exhibiting exencephaly (arrows) are each shown (center) together with a normal embryo from the same litter (bottom). Scale bars : 1 mm. ( C ) Distribution of mice at birth from the indicated crosses. Of note, out of five Trp53 YC/YC females observed at birth, only one remained alive at weaning age. Thus, the female/male ratio for weaned Trp53 YC/YC animals from these crosses was 1/22, a ratio similar to the one observed in A (4/75).

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques:

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A–B ) In homozygous males, p53 Y217C leads to accelerated tumor-induced death ( A ), and aggressive metastatic tumors ( B ); n=cohort size. ( C ) Hematoxylin and eosin (H&E) staining of sections from the lung (top) and spleen (bottom) of Trp53 -/- and Trp53 YC/YC male mice, showing metastases in Trp53 YC/YC animals. Normal organ structures are shown, with ‘A’ indicating pulmonary alveoli, and ‘WP’ and ‘RP’ standing for splenic white and red pulp, respectively. In the lung section of the Trp53 YC/YC mouse, the rectangle indicates a lymphoma area. In the spleen section of the Trp53 YC/YC mouse, the typical splenic structures are absent due to massive tissue homogenization of the spleen by lymphoma cells.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Staining, Tissue Homogenization

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Heat-map plot, with 717 differentially expressed genes suggestive of a loss of function (LOF), a separation of function (SOF), or a gain of function (GOF) for the p53 Y217C mutant, ranked according to log 2 fold change (n=number of genes). ( B ) Evidence of LOF in Trp53 YC/YC cells for genes encoding Puma, p21, and Zmat3. Data from three mice per genotype. Means + SEM are shown. ***p<0.001, **p<0.01, *p<0.05, °p=0.07, ns: non-significant by Student’s t-tests.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Mutagenesis

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: Effects of the Trp53 Y217C mutation: a summary. The comparison between Trp53 -/- and Trp53 Y217C/Y217C mice is presented. The phenotypes observed in Trp53 -/- male (M) and female (F) mice result from p53 loss of function (LOF), whereas those observed in Trp53 Y217C/Y217C mice result from p53 LOF as well as additional effects (gain of function [GOF] in bold). The + signs denote the presence of a phenotype. Xi: X chromosome inactivation.

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Mutagenesis, Comparison

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet:

Article Snippet: Cellular fractions were analyzed by western blots with antibodies against p53 (AF-1355, R&D Systems, 1/600), Tubulin (ab15568, Abcam, 1/1000), Nup98 (ab50610, Abcam, 1/1000), and histone H3 (ab1791, Abcam, 1/1000).

Techniques: Cell Culture, Sequencing, Recombinant, SYBR Green Assay, Staining, Software

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 is upregulated in HCC and associated with unfavorable prognosis. (A) Expression analysis of PDRG1 across multiple HCC datasets from the HCCDB database showing significant upregulation in tumor tissues compared with non-tumorous liver tissues. (B, C) Representative immunoblotting and IHC images of PDRG1 expression in paired HCC and adjacent noncancerous tissues from our cohort (n = 86). (D) The Cox regression analysis showed that in the public dataset, the higher the expression level of PDRG1, the worse the overall survival (OS), disease-free survival (DFS), disease specific survival (DSS), and progression-free survival (PFS). (E, F) OS and PFS analyses in our clinical cohort confirming that high PDRG1 expression predicts poor prognosis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 regulates proliferation, migration, invasion, and EMT in HCC cells. (A) Western blot validation of PDRG1 knockdown efficiency in HuH-7 and HLF cells and overexpression in SNU449 cells. (B) Wound-healing assays showing impaired migration upon PDRG1 knockdown and enhanced migration upon PDRG1 overexpression. (C) Transwell migration and invasion assays demonstrating reduced motility and invasiveness after PDRG1 knockdown and increased motility after PDRG1 overexpression. (D) Colony formation assays showing decreased clonogenicity upon PDRG1 knockdown and increased colony-forming ability with PDRG1 overexpression. (E) EdU incorporation assays indicating decreased proliferation in PDRG1-silenced cells and elevated proliferation in PDRG1-overexpressing cells. (F) Western blot analysis of EMT markers showing increased epithelial markers (E-cadherin and β-catenin) and decreased mesenchymal markers (N-cadherin and Vimentin) after PDRG1 knockdown, with the opposite effect upon PDRG1 overexpression. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Migration, Western Blot, Biomarker Discovery, Knockdown, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: Transcriptomic profiling reveals that PDRG1 suppresses tumor cell senescence and promotes HCC progression. (A) Volcano plot showing 696 differentially expressed genes upon PDRG1 overexpression. (B) Heatmap of the top significantly altered genes in PDRG1-overexpressing cells. (C) GSEA showing enrichment of senescence-related signatures in PDRG1-overexpressing cells. (D) Western blot analysis showing that PDRG1 knockout increases p21 and SASP factors (IL-6, IL-8, and IL-1β) while decreasing p-CDK2 and p-RB; conversely, PDRG1 overexpression reduces p21 and SASP factors and increases p-CDK2 and p-RB. (E) Xenograft assays show that SNU449 cells overexpressing PDRG1 display enhanced tumor growth, as evidenced by increased tumor volume and tumor weight. (F-H) IHC analysis of Ki-67, p-RB, p21, IL-6, and IL-1β in xenograft tumors showing reduced senescence-associated markers in PDRG1-overexpressing tumors. (I) CCK-8 assays showing that p21 restoration significantly attenuates proliferation in PDRG1-overexpressing SNU449 cells. (J) EdU incorporation assays showing that p21 restoration reduces DNA synthesis in PDRG1-overexpressing SNU449 cells. (K) Wound-healing assays showing that p21 overexpression reverses the enhanced migratory capacity induced by PDRG1 overexpression. (L) Transwell migration and invasion assays showing that p21 restoration suppresses PDRG1-induced increases in migration and invasion. (M) Colony formation assays showing that p21 overexpression markedly reduces clonogenic growth in PDRG1-overexpressing SNU449 cells. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Over Expression, Western Blot, Knock-Out, CCK-8 Assay, DNA Synthesis, Migration

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 interacts with EZH2 and positively regulates EZH2 expression in HCC. (A) IHC staining showing elevated EZH2 expression in HCC tissues compared with adjacent non-tumor tissues (n = 86). (B) Correlation analysis of PDRG1 and EZH2 protein expression in clinical HCC samples. (C) TCGA-LIHC dataset confirming the positive correlation between PDRG1 and EZH2 mRNA expression. (D) Co-IP assays showing direct binding between PDRG1 and EZH2 in HLF cells. (E) Western blot showing that PDRG1 knockdown reduces EZH2 protein levels, whereas EZH2 overexpression does not affect PDRG1 expression. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Expressing, Immunohistochemistry, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Knockdown, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: EZH2 restores malignant phenotypes suppressed by PDRG1 knockdown. (A, B) CCK-8 assays showing that EZH2 overexpression restores cell proliferation in PDRG1-silenced HLF and HuH-7 cells. (C, D) Wound-healing assays showing that EZH2 overexpression rescues the migration defects induced by PDRG1 knockdown. (E, F) Transwell assays demonstrating that EZH2 overexpression reverses PDRG1 knockdown-mediated suppression of migration and invasion. (G, H) Colony formation assays showing increased clonogenicity upon EZH2 overexpression in PDRG1-silenced cells. (I, J) EdU assays showing that EZH2 overexpression restores reduced DNA synthesis in PDRG1-knockdown cells. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Knockdown, CCK-8 Assay, Over Expression, Migration, DNA Synthesis

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 regulates p21 transcription through EZH2-mediated H3K27me3 modification. (A) Western blot analysis showing that PDRG1 knockdown decreases EZH2, p-CDK2, and p-RB levels while increasing p21 and SASP factors (IL-1β, IL-6, and IL-8); ectopic EZH2 expression reverses these changes. (B, C) ChIP-qPCR analysis showing that EZH2 knockdown reduces, whereas EZH2 overexpression increases, EZH2 occupancy and H3K27me3 enrichment at the p21 promoter in HLF cells. (D, E) ChIP-qPCR analysis demonstrating that PDRG1 knockdown decreases EZH2 recruitment and H3K27me3 levels at the p21 promoter, both of which are restored by EZH2 re-expression in HLF cells. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Modification, Western Blot, Knockdown, Expressing, ChIP-qPCR, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: Structural identification of the PDRG1-EZH2 binding interfaces and the functional requirement of their interaction in HCC progression. (A) Co-IP using EZH2 and PDRG1 truncation mutants demonstrates that the N-terminal region of EZH2 (amino acids 1-340) and the N-terminal segment of PDRG1 (amino acids 36-70) mediate their interaction in SNU449 cells. (B) Structural modeling predicts a stable interaction interface between the N-terminal helix of PDRG1 and the N-terminal region of EZH2. (C-F) CCK-8, wound-healing, Transwell, and colony formation assays show that overexpression of the PDRG1 Δ36-70 promotes cell proliferation, migration, invasion, and clonogenicity in SNU449 cells. All experiments were performed with three independent biological replicates (n = 3). (G) Western blot analysis indicates that the overexpression of PDRG1 Δ36-70 suppresses p21 and SASP factors and activates p-CDK2 and p-RB in SNU449 cells. (H) Xenograft assays show that SNU449 cells expressing PDRG1 Δ36-70 exhibit enhanced tumor growth, as indicated by increased tumor volume and tumor weight (n = 6). oePDRG1 PD2 : oePDRG1 Δ36-70 , * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Binding Assay, Functional Assay, Co-Immunoprecipitation Assay, CCK-8 Assay, Over Expression, Migration, Western Blot, Expressing

Journal: Antioxidants

Article Title: Liproxstatin-1 Attenuates Retinal Ischemia–Reperfusion Injury by Suppressing EGR1-Mediated Ferroptosis

doi: 10.3390/antiox15030391

Figure Lengend Snippet: Lip-1 halts the oxidative stress-to-ferroptosis cascade in retinal I/R injury. ( A ) Immunofluorescence co-staining of Tuj1-positive RGCs (green) with GPX4 (red), FSP1 (red), and ACSL4 (red) and DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( B ) Immunofluorescence co-staining of Tuj1-positive RGCs (green) with 4HNE (red), FHC (red), and DHE (red) and DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( C ) Immunofluorescence co-staining of FerroOrange (red) with DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( D ) Quantification of the relative immunofluorescence intensity of GPX4, FSP1, ACSL4, 4-HNE, FHC, DHE, and FerroOrange from ( A – C ) ( n = 6 biologically independent experiments). ( E ) Immunofluorescence co-staining of C11-BODIPY (green/red) staining with DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( F ) Quantification of the C11 fluorescence ratio (green/red) from ( E ) ( n = 6 biologically independent experiments). ( G ) Western blot analysis of GPX4, FSP1, and FHC expression from sham and I/R mice treated with Vehicle or Lip-1. β-Actin served as a loading control. ( H ) Quantification of GPX4, FSP1, and FHC protein levels from ( E ) ( n = 3 biologically independent experiments). Scale bars: 50 μm. Total magnification: 400×. Data were analyzed by two-way ANOVA with Tukey’s post hoc test. All data are shown as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: FSP1 , Rabbit , Proteintech , 20886-AP , 1:1000 (WB).

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Control

Journal: Antioxidants

Article Title: Liproxstatin-1 Attenuates Retinal Ischemia–Reperfusion Injury by Suppressing EGR1-Mediated Ferroptosis

doi: 10.3390/antiox15030391

Figure Lengend Snippet: Lip-1 attenuates OGD/R-induced ferroptosis in primary RGCs by restoring antioxidant defenses and suppressing lipid peroxidation. ( A ) Western blot analysis of GPX4 expression in primary RGCs at the indicated time points (3, 6, 12, 24 h) after OGD/R. β-Actin served as a loading control. ( B ) Quantification of GPX4 relative band density from ( A ) ( n = 3 biologically independent experiments). ( C ) Western blot analysis of GPX4, FSP1, and FHC expression in RGCs under different conditions (Control, OGD/R + Vehicle, OGD/R + Lip-1). β-Actin served as a loading control. ( D ) Quantification of GPX4, FSP1, and FHC protein levels from ( C ) ( n = 3 biologically independent experiments). ( E ) Immunofluorescence co-staining of Tuj1-positive RGCs (red) with GPX4 (green) and DAPI (blue) under different conditions. ( F ) Immunofluorescence co-staining of Tuj1-positive RGCs (red) with ferroptosis markers (FSP1, ACSL4, 4-HNE, FHC, all in green) and DAPI (blue) under different conditions. ( G ) Immunofluorescence co-staining of Tuj1-positive RGCs (purple) with FerroOrange staining (red), DCFH-DA (green) and DAPI (blue) under different conditions. ( H ) Quantification of the relative immunofluorescence intensity of GPX4, FSP1, ACSL4, 4-HNE, FHC, FerroOrange, and DCFH-DA from ( E – G ) ( n = 6 biologically independent experiments). ( I ) Immunofluorescence co-staining of Tuj1-positive RGCs (purple) with lipid peroxidation (detected by oxidized C11-BODIPY581/591, green/red), mitochondrial membrane potential (assessed by JC-1 monomer/aggregate ratio, green/red) and DAPI (blue) under different conditions. ( J ) Quantification of the C11 and JC-1 fluorescence ratio (green/red) from ( I ) ( n = 6 biologically independent experiments). ( E – G , I ) Scale bars: 50 μm. Total magnification: 400×. Data were analyzed by two-way ANOVA with Tukey’s post hoc test. All data are shown as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: FSP1 , Rabbit , Proteintech , 20886-AP , 1:1000 (WB).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Membrane, Fluorescence