Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 1 Silencing Arg-II in senescent cells inhibits eNOS uncoupling, reverses endothelial senescent phenotypic changes, and suppresses endothelial inflammation. Senescent human umbilical vein endothelial cells (HUVECs) were transduced with rAd ⁄ U6-LacZshRNA as control (con) or rAd ⁄ U6-Arg-IIshRNA to silence Arg-II gene. (A) Immunoblotting shows Arg-II silencing in senescent cells. (B) DHE staining for detection of O 2 and DAF-2DA staining for detection of NO. Quantifications of DHE and DAF-2DA signals are shown below. (C) SA-b-gal staining. Bar graphs show quantifications of SA-b-gal-positive cells. (D) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Transduction, Control, Western Blot, Staining

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 2 Co-expression of superoxide dismutase-1 (SOD1) in young endothelial cells prevents Arg-II-induced eNOS-uncoupling, endothelial senescence and inflammation. Young endothelial cells were transduced with empty rAd ⁄ CMV vector as control (con) or rAd ⁄ CMV-Arg-II alone or rAd ⁄ CMV-Arg-II plus rAd ⁄ CMV-SOD1. (A) Immunoblotting analysis to confirm overexpression of Arg-II and SOD1. (B) DHE staining for detection of O 2 and DAF-2DA staining for detection of NO and effect of SOD1. Bar graphs show quantifications of DHE and DAF-2DA signals. (C) SA-b-gal staining and effect of SOD1. Bar graphs show quantifications of percentage of SA-b-gal positive cells. (D) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1 levels, and endothelial inflammation markers vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) expression. Tubulin served as loading control. Bar graphs show quantifications of the markers. (E) CFDA-SE fluorescence labeled THP-1 monocyte adhesion to endothelial cells that were transduced with rAd expressing transgenes as indicated. Bar graphs show quantifications of the adhered monocytes. *P < 0.05, **P < 0.01 and ***P < 0.005 vs. control (con); ††<0.01 and †††P < 0.005 vs. Arg-II. Scale bar = 0.2 mm.

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Western Blot, Over Expression, Staining, Labeling

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 5 Silencing Arg-II prevents S6K1-induced eNOS-uncoupling, senescence and inflammation in young endothelial cells. The transduction procedure of young cells was the same as in Fig. 5A. (A) DHE staining for detection of O 2 and DAF-2DA staining for detection of NO and effect of Arg-II silencing. Bar graphs show quantifications of DHE and DAF-2DA signals. (B) SA-b-gal staining and effect of Arg-II silencing. Bar graphs show quantifications of percentage of SA-b-gal positive cells. (C) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1 levels, and endothelial inflammation markers vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1). Tubulin served as loading control. Bar graphs show quantifications of the markers. (D) CFDA-SE fluorescence labeled THP-1 monocyte adhesion to endothelial cells that were transduced with rAd expressing transgenes and shRNA as indicated. Bar graphs show quantifications of the adhered monocytes. **P < 0.01, ***P < 0.005 vs. control (con ⁄ LacZ); †P < 0.05, ††P < 0.01, †††P < 0.005 vs. S6K1ca ⁄ LacZ group. Scale bar = 0.2 mm.

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Transduction, Staining, Western Blot, Control, Labeling, Expressing, shRNA

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 6 Deficiency in Arg-II gene in mice (Arg-II) ⁄ )) protects against vascular inflammation and aging. Aortas of young (2–3 months) and old (23–24 months) wild-type (WT) and Arg-II) ⁄ ) mice were cleaned of perivascular tissues and subjected to en face staining or Immunoblotting analysis. (A) qRT-PCR analysis of Arg-II mRNA levels. (B) Confocal microscopic en face detection of endothelial vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), vWF (the endothelial marker), followed by counterstaining with DAPI. Shown are representative images of each group. (C) Immunoblotting analyses of VCAM1, ICAM1, p21 levels in the aortas and p53-Ser15 and p53 levels in the heart of young and old WT and Arg-II) ⁄ ) mice. (D and E) Quantifications of the above results. Tubulin is taken as loading control. n = 4 mice in each group. **P < 0.01, ***P < 0.001 vs. young WT mice; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. old WT mice. Scale bar = 100 lm.

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Marker, Control

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Clinical characteristics of the patients with p53 staining patterns.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Staining, Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Recurrence patterns involving both p53 mutant and wild-type patterns.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Overall survival and recurrence-free survival of patients with both the p53 wild-type and mutant patterns. Kaplan-Meier curves for (A) overall survival and (B) recurrence-free survival over a period of 5 years with the p53 wild-type and p53 mutant pattern. Patients with the p53 mutant pattern had low (A) overall survival and (B) recurrence-free survival rates in all patients. In subgroup analysis, the recurrence-free survival rate was lower in patients with the p53 mutant pattern than in those with the wild-type pattern as regards both (C) pN0 or (D) pN+, and (E) early and (F) advanced-stage gastric cancer.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Comparison of the characteristics of patients with early- and advanced-stage gastric cancer in association with the p53 wild-type and p53 mutant pattern.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Comparison, Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Clinical characteristics of the patients with p53 staining patterns before and after matching on the propensity score.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Staining, Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Comparison of patient characteristics between pN0 and pN+ gastric cancer in p53 wild-type and mutant pattern.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Comparison, Mutagenesis

Journal: Journal of cellular and molecular medicine

Article Title: Functional role of RRS1 in breast cancer cell proliferation.

doi: 10.1111/jcmm.13922

Figure Lengend Snippet: FIGURE 5 P53 and p21 are induced by RRS1 knockdown. MCF‐ 7 cells were infected with a retrovirus expressing RRS1 (shRRS1) or with a Ctrl vector (shctrl) for 2 days. Whole‐cell lysates were analysed by Western blot. P53 and p21 expression levels were increased by RRS1 knockdown (*P < 0.05 vs shctrl)

Article Snippet: For western blotting, xenograft tumors and cell lines were lysed, and protein samples were harvested as previously described.29 Equal amounts of protein were resolved by SDS‐PAGE and blotted using antibodies specific to RRS1 (1:1000, Abcam), p53 (1:500, OriGene), RPL11 (1:1000, Abcam) and β‐actin (1:1000, Bioss, Beijing, China).

Techniques: Knockdown, Infection, Expressing, Plasmid Preparation, Western Blot

Journal: Archives of Biological Sciences

Article Title: Quercetin and lithium chloride modulate Wnt signaling in pluripotent embryonal carcinoma NT2/D1 cells

doi: 10.2298/abs1301201m

Figure Lengend Snippet: Fig. 2. LiCl decreases the proliferation rate of NT2/D1 cells in a p53-dependent manner. (A) MTT proliferation assay of NT2/D1 treated with lithium chloride-7, 10 and 20 mM, for 24 h. The results are shown as per- centages of the negative control, untreated NT2/D1 cells. Values are presented as the means ±S.E.M. of at least three independent experiments. Mean values of relative proliferation rates were compared using Student’s t test. Values of p<0, 05 are presented by *. (B) Western blot analysis of p53 protein expression in NT2/ D1 cells treated with 7, 10 and 20 mM lithium chloride for 24 h. The level of GAPDH was used as a control for equal amounts of input proteins.

Article Snippet: Western blots were performed using anti c-myc (9E10) (Santa Cruz Biotechnology), anti-p53 (DO1) (Gene Spin), anti α-tubulin (DM1A) (Calbi- ochem), and anti-GAPDH (AM20337PU-S) (Acris Antibodies, Inc).

Techniques: Proliferation Assay, Negative Control, Western Blot, Expressing, Control

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: 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: Communications biology

Article Title: Assessing phototoxic drug properties of hydrochlorothiazide using human skin biopsies.

doi: 10.1038/s42003-025-08064-1

Figure Lengend Snippet: Fig. 2 | Low dose UVA irradiation caused increased p53 protein levels and enhanced phos- phorylation and MDM2 mRNA expression in HCT-treated skin biopsies independent of γH2A.X. A Representative Western blots demon- strating protein level of tumor suppressor protein p53, phosphorylation of histone H2A.X (γH2A.X, Serin139) and phosphorylation of p53 (Serin15) in untreated control biopsies (Ctrl), and in biopsies treated with HCT 6 h after irradiation with 300 mJ/ cm2 UVA or UVB. Unirradiated biopsies served as group-specific control (non). Protein expression of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) served as loading control. Quantification of (B) p53 protein (C) phospho-p53, and (D) γH2A.X. E Gene expression of p53-regulator MDM2 normalized against GAPDH 6 h after irra- diation. F Representative Western blots of p53, γH2A.X and phospho-p53 (Serin15) in untreated control biopsies (Ctrl), and in biopsies treated with HCT 24 h after irradiation with 300 mJ/cm2 UVA or UVB. Unirradiated biopsies served as group-specific control (non). Protein expression of GAPDH served as loading control. Quantification of (G) p53 protein (H) phospho-p53, and (I) γH2A.X. J Gene expres- sion of MDM2 normalized against GAPDH 24 h after irradiation. For (B, C, D, E, G, H, I, J) n = 6 biopsies per group. Data are shown as mean ± SEM with individual points. For comparison of three groups, P-value was determined using Kruskal- Wallis with Dunn´s multiple comparisons test for Ctrl group in (B, C, D, G, H, I) and One-way ANOVA with Tukey multiple comparisons test for HCT group in (B, C, D, G, H, I). For (E and J) One- way ANOVA with Tukey multiple comparisons test was used. An unpaired student t-test (#) was used for comparison of 2 groups for (B, C, E, G, H, J). IOD: Integrated optical density. Numerical source data are provided within the Supplementary Data 1 file.

Article Snippet: Slices were incubated overnight with primary antibody (Anti-p53 (DO-7) mouse mAb, (#48818S; Cell Signaling Technologies), diluted (1:100) in 1xPBS in a moisture-chamber at +4 °C.

Techniques: Irradiation, Expressing, Western Blot, Phospho-proteomics, Control, Gene Expression, Comparison