Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: Molecular characteristics of different p53 statuses of colorectal carcinoma in TCGA. (A) Genomic mutation signature in the patients with colorectal carcinoma (COAD) from TCGA database. The upper panel shows the mutation percentage of p53 in all COAD samples. The middle panel shows the mutation percentage of p53 in tumor-free samples. The bottom panel shows the mutation percentage of p53 in with-tumor samples. (B) Gene set enrichment in mutant p53 and wild-type p53 groups, respectively ( p < 0.05, FDR < 0.25).

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: Differences of clinical pathological characteristics between missense p53 and wild-type p53 groups.

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: Cell growth, migration, invasion, and stemness in HCT116 with different p53 statuses. (A) Images of Western blot for p53 in HCT116 p53 (+/+), p53 (-/-), p53 (R273H), and p53 (R248W). RPS18 was used as internal control. (B) Growth rate of the different statuses of p53 in HCT116. (C) Images of colonogenic formation in HCT116-derived cell lines with different p53 status. (D–F) . Images of tumorsphere formation, migration, and invasion in each HCT116-derived cell lines. * p < 0.05, ** p < 0.01, *** p < 0.001, ****, and p < 0.0001. The error bar was from three independent experiments.

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Migration, Western Blot, Control, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: Anti-apoptosis and 5-FU resistance in HCT116 with different p53 statuses. (A) Representative images for live-dead staining in HCT116-derived cell lines. Red dot represents dead cells, and green dot shows alive cells. (B) Representative images for FITC-Annexin V/PI flow cytometry in HCT116-derived cell lines. The areas of Q2 and Q3 are considered as apoptotic cells. (C) The expressions of cleaved-caspase3 and cleaved PARP protein in HCT116-derived cell lines. (D) mRNA and protein levels of TYMS in different HCT116-derived cell lines. **** p < 0.0001. The error bar was from three independent experiments.

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Staining, Derivative Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: Expression levels of CD44 in different HCT116 cell lines. (A,B) mRNA and protein levels of CD44 in HCT116 p53 (+/+), p53 (-/-), p53 (R273H), and p53 (R248W). RPS18 was internal control. (C,D) . Images for CD44-positive cells in HCT116-derived cell lines. * p < 0.05. The error bar was from three independent experiments.

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Expressing, Control, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: CD44 knockdown enhanced chemosensitivity in different HCT116 cell lines. (A) Western blot detection for the knockdown efficiency of three siCD44s in HCT116 p53 (+/+) and HCT116 53 (-/-) cells. siNC serves as the negative control. RPS18 was used as internal control of Western blot. (B) Protein levels of TYMS and CD44 in different HCT116 cell lines after knockdown of CD44 by siRNA. (C) Images of tumorsphere formation in CD44 knockdown HCT116 cell lines (magnification 400×). (D) Representative images for FITC-Annexin V/PI flow cytometry in siCD44-transfected HCT116 cell lines. The cells in Q2 and Q3 are considered as apoptotic cells. The error bar was from three independent experiments. (ns, no significance; ** p < 0.01, **** p < 0.0001).

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Knockdown, Western Blot, Negative Control, Control, Flow Cytometry, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: In vivo chemoresistance of deficient and mutant p53 HCT116 cell lines. (A) Tumor size of different HCT116-derived cell lines at 10 days post-transplantation in the right back of the mice. (B) Tumor growth curve of different HCT116-derived cell lines under 5-FU administration. (C) Immunohistochemistry scores of p53, CD44, and TYMS expressions in tumors. (D) Representative images for IHC staining of p53, CD44, and TYMS in tumor species. Brown areas show positive tumor cells. p53 almost located in nucleus, CD44, and TYMS are expressed in cytoplasm. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: In Vivo, Mutagenesis, Derivative Assay, Transplantation Assay, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: p53/TCF21/CD44 axis promotes chemoresistance in deficient and mutant p53 HCT116-derived cell lines. (A) Diagram for the luciferase reporter with the CD44 promoter, and two putative binding sites of TCF21 were deleted when the pGL3B-CD44mut plasmid was constructed. (B) Fold changes in luciferase activities of pGL3B-CD44wt with TCF21 and pGL3B-CD44mut without TCF21 binding sites in each HCT116-derived cell lines. (C,D) mRNA and protein levels in different HCT116 cells. RPS18 was performed as internal control. (E–G) . The expression changes at mRNA and protein levels in siCD44-transfected HCT116-derived cell lines. The error bar was from three independent experiments. (ns, no significance; **** p < 0.0001).

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Mutagenesis, Derivative Assay, Luciferase, Binding Assay, Plasmid Preparation, Construct, Control, Expressing, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: TCF21 overexpression rescues chemoresistance in deficient and mutant p53 HCT116 cell lines. (A) Images of tumorsphere formation in TCF21 overexpressed HCT116-derived cell lines (magnification ×400). (B) Protein levels of TYMS, CD44, and TCF21 in different HCT116-derived cell lines. RPS18 was internal control. (C) . Representative images for FITC-Annexin V/PI flow cytometry in TCF21 overexpressed HCT116-derived cell lines. The cells in Q2 and Q3 are considered as apoptotic cells. The error bar was from three independent experiments (ns, no significance; ** p < 0.01, **** p < 0.0001).

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Over Expression, Mutagenesis, Derivative Assay, Control, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

doi: 10.3389/fcell.2021.788331

Figure Lengend Snippet: Scheme of the novel mechanism by which p53 regulates chemoresistance to 5-FU via TCF21/CD44 axis-mediated enhanced stemness in colorectal carcinoma. TCF21 is rich in tumor cells with wtp53 and can directly bind to the CD44 promoter to suppress the expression levels of CD44, leading to the decrease of TYMS. In contrast, CD44 is abundant in tumor cells with mutp53 because of a low level of TCF21, resulting in the increase of TYMS, thus enhancing chemoresistance, stemness, and proliferation of colorectal carcinoma cells.

Article Snippet: The p53-null HCT116 p53 (-/-) cells were transduced HCT116 stable expressing p53 R273H and p53 R248W cell lines and were generated by transduction with pCMV-Neo-Bam mutp53 plasmids expressing mutant p53 with R273H or R248W into HCT116 p53 (-/-) cells (pCMV-Neo-Bam-p53 R273H and R248 mutation vectors were a gift from Dr. Bert Vogelstein (Addgene plasmids #16437 and #16439), G418 selection and single colony screening, respectively.

Techniques: Expressing

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: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 1. Combined treatment for 4 h with EPA-FFA, EGCG and GS in CRC cells had effects on mTOR pathway. (A) HCT116 cells were treated with EPA- FFA (0–150 µM), EGCG (0–175 µM), GS (0–15 µM) and the number of viable cells compared with the control (%) was assessed with MTT assay (ANOVA P = 0.0016, **P < 0.01 Dunnett’s test, n = 3) (left). mTOR downstream targets P-p70S6K, p70S6K, P-4EBP1, 4EBP1 were detected by western blotting on cells treated with compounds alone or in combinations or with Rapamycin. Statistical significance was tested only on HCT116 cells treated with EPA-FFA 150 µM, EGCG 175 µM, GS 15 µM or Rapamycin compared with the control (untreated cells) using one-way ANOVA (P = 0.0285 for P-p70S6K on logarithmic transformed data and P = 0.0347 for P-4EBP1) followed by Tukey’s test, n = 4 (right). (B) SW480 cells were treated with EPA-FFA, EGCG and GS and the number of viable cells compared with the control was evaluated with MTT assay (ANOVA P = 0.0004, *P < 0.05; ***P < 0.001 Dunnett’s test, n = 3) (left). mTOR downstream targets P-p70S6K, p70S6K, P-4EBP1, 4EBP1 were assessed upon treatment. Statistical significance was tested on SW480 cells treated with EPA-FFA 150 µM, EGCG 175 µM, GS 15 µM or Rapamycin 20 nM compared with the control (untreated cells) using one-way ANOVA (P = 0.0294 for P-p70S6K on logarithmic transformed data and P = 0.0008 for P-4EBP1) followed by Tukey’s test, n = 4 (right).

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Control, MTT Assay, Western Blot, Transformation Assay

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 2. Effect of EPA-FFA, EGCG and GS on mRNA translation in HCT116 cells. (A) Polysomal profiles on control and treated cells are shown. Statistical significance was assessed using unpaired t-test (P = n.s., n = 2) (B) mRNA levels of L5, L11, L13, c-Myc and cyclin D1 were assessed by qRT-PCR both in total RNA and in polysomal associated RNA. Statistical significance was assessed using one sample t-test, n = 2, using 100 (control) as the reference value.

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Control, Quantitative RT-PCR

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 3. Effect of EPA-FFA, EGCG and GS on c-Myc and cyclin D1. c-Myc and cyclin D1 proteins levels were assessed in cell extracts treated with EPA- FFA+EGCG+GS or Rapamycin from (A) HCT116 or (B) SW480 cells. Analyses were performed on logarithmic transformed data for HCT116. After the ANOVA global test (P = 0.0190, n = 4, HCT116; P = n.s., n = 4), Tukey’s post hoc test was used for pairwise comparisons.

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Transformation Assay

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 4. Effect of EPA-FFA, EGCG and GS on cell proliferation and apoptosis in HCT116 and SW480 cells. (A) Clonogenic assay on HCT116 and SW480 cells treated with EPA-FFA+EGCG+GS or Rapamycin (ANOVA P = 0.0012 and P = 0.0017 for HCT116 and SW480, respectively; Tukey’s test was applied as post hoc test, n = 3). (B) Cell cycle analysis (ANOVA P = 0.0059 for HCT116 and P = n.s. for SW480, n = 3, Dunnett’s test) (C) Tunnel assay: representative pictures and quantification. Statistical significance was tested using one-way ANOVA (P = 0.0447 for HCT116 and P = 0.0011 for SW480, followed by Dunnett’s test for comparison with the control cells, n = 2). Ctrl = control; Comb = Combination; Rapa = Rapamycin.

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Clonogenic Assay, Cell Cycle Assay, Comparison, Control

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: mTOR activity and metabolic differences in U251, U87 and U373-U human glioma cells. a mTOR activity related proteins and other metabolic enzyme expressions characterise and show some individual differences in the studied human glioma cell lines—representative figures of Western blot results; b the enzyme expression profiles could correlate to mTOR inhibitor sensitivity of glioma cells (rapamycin—Rapa 50 ng/mL; NVP-BEZ235—BEZ 1 µM; PP242 1 µM for 72-h treatments), which were monitored by Alamar Blue and SRB proliferation tests—the cell proliferation of untreated controls was considered 100%; rapamycin inhibited the proliferation in all studied cells, significantly; Significant differences compared to rapamycin were labelled by *p < 0.05

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Western Blot, Expressing

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: The anti-proliferative effects and alterations in mTOR activity and other metabolism related protein levels in response to mTORI and temozolomide treatments in human glioma cell lines. a mTOR inhibitors (rapamycin—Rapa 50 ng/mL; NVP-BEZ235—BEZ 1 µM; PP242 1 µM) and temozolomide (TMZ 100 µM) 72-h combination treatments effectively inhibited the proliferation of U251, U87 and U373-U cells—Alamar Blue and SRB proliferation test results, the cell proliferation of untreated controls were considered 100% (temozolomide slightly reduced the proliferation and had no significant growth inhibitory effect on U251, mTORIs had similar effects as in Fig. a, and all temozolomide + mTORI combined treatments had significant anti-proliferative effects, p < 0.05; the additive (A) or synergistic (S) effects of inhibitor combinations were given based on CI calculation, SD was added); b altered expressions of mTORC1 and C2 activity related proteins and other metabolic enzymes were also shown after different mono-treatments (results of representative Western blots after 72-h treatments)

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Western Blot

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: Metabolic drugs induced proliferative and protein expression profile changes in human glioma cells. a Doxycycline (Doxy 10 µM), etomoxir (Etom 50 µM), chloroquine (Chl 50 µM) 72-h treatments have slight growth inhibitory effects in U251, U87 and U373-U (Alamar Blue proliferation test data; *p < 0.05); b ACSS2, FASN, CPT1a and p-(Ser473)-Akt protein expressions were influenced by rapamycin, doxycycline, etomoxir and chloroquine after 72-h mono-treatments; c FASN, β-F1-ATPase, p-S6, p-(Ser473)-Akt and CPT1a protein expression compared to the untreated controls after applying two-drug combinations in U251 glioma cells (50 ng/mL rapamycin + 100 µM temozolomide—Rapa + TMZ; 50 ng/mL rapamycin + 10 µM doxycycline—Rapa + Doxy; 100 µM temozolomide + 10 µM doxycycline—TMZ + Doxy (Western blot results)—representative Western blot figures

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Western Blot

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: Rapamycin and temozolomide combined with other metabolic inhibitors have different in vitro anti-proliferative effects in human glioma cell lines. Alamar Blue test results after 72-h different two-drug combined in vitro treatments in U251 ( a ), U87 ( b ) and U373-U ( c ) glioma cells (rapamycin—Rapa 50 ng/mL, temozolomide—TMZ 100 µM, doxycycline—Doxy 10 µM; etomoxir—Etom 50 µM; chloroquine—Chl 50 µM). The additive (A) or synergistic (S) effects of combinations were given based on CI calculation, SD was added, the anti-proliferative effects were significant (p < 0.05) in almost all combined treatments compared to untreated cultures except for doxycycline + etomoxir and temozolomide + etomoxir treatments

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vitro

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: miR-26b enhances HCC cell sensitivity to doxorubicin. A. QRT-PCR analysis of the changes in miRNA after treatment with doxorubicin in SNU449 and SNU387 cells. B. The level of miR-26b was determined following treatment with or without Doxorubicin by qRT-PCR in HCC cells. **P < 0.01. C. A CCK-8 assay analysis showed that treatment with an miR-26b mimic can enhance the sensitivity of HCC cells to doxorubicin, with the exception of Hep3B cells. D and E. An EdU incorporation assay of cellular proliferation in different treatment groups. **P < 0.01. F and G. The apoptosis ratio was determined by flow cytometry. *P < 0.05; **P < 0.01. H. QRT-PCR used to determine miR-26b expression in adjacent cancer and adjacent tissues. I. We used StarBase v 3.0 project to analyze the level of miR-26b in normal and cancer tissues.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques: Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Expressing

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: p53 was related to the sensitivity of HCC cells to doxorubicin. A. P53 protein expression was detected by Western blot. B - E. The cell viability was examined in doxorubicin, or miR-26b mimic plus doxorubicin treated cells following transfection with p53 siRNA.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques: Expressing, Western Blot, Transfection

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: miR-26b enhances HCC cell sensitivity to doxorubicin via USP9X-dependent p53 degradation. A. Western blot indicating the expression of p53 after treated with doxorubicin, doxorubicin plus miR-26 mimic, or MG132. **P < 0.01, ***P < 0.001. B. Tenovin-1 combined with an miR-26b mimic could enhance doxorubicin sensitivity in HepG2 cells, except other HCC cells. C. The predicted miR-26b binding site in the USP9X 3′ UTR and dual fluorescence reporter gene experiments verify that miR-26b binds to the promoter region of USP9X. *P < 0.05, **P < 0.01, ***P < 0.001. D. The expression of USP9X was up-regulated following treatment with the miR-26b mimic. *P < 0.05, **P < 0.01. E. P53 protein expression was determined by Western blot. F. The expression of UXP9X was determined by Western Blot.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques: Western Blot, Expressing, Binding Assay, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: miR-26b enhances doxorubicin sensitivity via autophagy. A. The expression of proteins in HCC cells in different treatment groups detected by Western blot. * P < 0.05, ** P < 0.01. B-D. Confocal microscopy analysis of LC3 double fluorescent cells. *P < 0.05; **P < 0.01; ***P < 0.001. E-F. Electron microscopy detected autophagic flow in SNU449 and HepG2 cells. **P < 0.01 G. CCK-8 detected the sensitivity to doxorubicin in HCC cells after treatment with different conditions (transfected with miR-26b mimics, miR-26b mimic interference, and treated with rapamycin). *P < 0.05 vs Negative Control.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques: Expressing, Western Blot, Confocal Microscopy, Electron Microscopy, CCK-8 Assay, Transfection, Negative Control

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: Treatment with Sp-94dr/miR-26b mimic combined with doxorubicin down-regulates p53 and USP9X expression in vivo . A and B . Immunohistochemistry-positive (IHC-P) cells were examined for the expression of USP9X and p53. *P < 0.05; **P < 0.01; ***P < 0.001. C. QRT-PCR analysis the expression of miR-26b, USP9X, and p53 in control, doxorubicin, sp-94dr/miR-26b mimic, and doxorubicin plus with sp-94dr/miR-26b mimic-treated cells. *P < 0.05; **P < 0.01; ***P < 0.001. D. USP9X, p53, and p62 protein expression were evaluated using a Western blot. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques: Expressing, In Vivo, Immunohistochemistry, Paraffin-embedded Immunohistochemistry, Quantitative RT-PCR, Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: Schematic diagram of the regulatory mechanism of the miR-26b/USP9X/p53 axis in regulating HCC sensitivity to doxorubicin.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

doi: 10.7150/ijbs.52517

Figure Lengend Snippet: IC 50 values for doxorubicin in HCC cell lines with or without miR-26b mimic siRNA treatment.

Article Snippet: Human HCC cells (HepG2 (Wild-type p53), Hep3B (p53 deletion) SNU387 and SNU449 (p53 mutation) were supplemented from ATCC.

Techniques:

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Inducible clonal T47D cells with mdm2 shRNA or control vector were treated with or without 4μg/ml doxycycline (dox) for 3 days, followed by 10nM estrogen for 5 days in the presence or absence of dox. A representative image of western blot analysis of MDM2, phospho Rb, E2F1, p53 and Actin protein levels from 50μg whole cell protein extract is shown. (B) ImageJ analysis was performed for MDM2, phospho Rb and E2F1 protein levels normalized to Actin. The graph represents an average of three independent experiments with standard deviation in inducible clonal T47D cells with mdm2 shRNA or control vector. (C) A constitutive pool of T47D cells with mdm2 shRNA or control vector were grown with or without 10nM estrogen for 5 days. A representative image of western blot analysis of MDM2, phospho Rb, E2F1, total Rb and Actin protein levels from 50μg whole cell protein extract is shown. (D) ImageJ analysis was performed for MDM2, phospho Rb and E2F1 protein levels normalized to Actin. Graph represents average of three independent experiments with standard deviation in constitutive pool of T47D cells with mdm2 shRNA or control vector. * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01, *** represents a p-value ≤ 0.001. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Control, Plasmid Preparation, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) T47D.sh mdm2 cells were treated with 10nM estrogen (lane 1) and either had MDM2 knockdown (lane 2) or 10μM fulvestrant treatment (lane 3), or both (lane 4) for 5 days. A representative western blot analysis of MDM2, phosphoRb and Actin protein levels from 50μg whole cell protein extract is shown. Dot plot diagram shows quantified ImageJ values of phospho Rb protein levels normalized to Actin from three independent experiments. (B) MTT assay was performed in T47D.control vector and inducible T47D.sh mdm2 clonal cell lines after treatments. Percentage mitochondrial activity represents an average of 2 independent experiments. (A) & (B) * represents a p-value ≤ 0.05, *** represents a p-value ≤ 0.001, **** represents a p-value ≤ 0.0001. The p-value was determined by 2-tailed Student t-test. (C) A representative live cell image by confocal microscopy with 20X objective of T47D.vector and inducible clonal T47D.sh mdm2 clonal cell lines after treatments. Red fluorescence represents staining with propidium iodide. Blue fluorescence represents staining of nuclear DNA. (D) MDM2 drives phosphorylation of Rb in ER+ breast cancer cells. In vitro kinase assay was performed to detect phosphorylation of Rb with or without overnight estrogen treatment in either presence or absence of bacterially expressed and purified MDM2 (1μl or 2μl). A representative image of Western blot analysis of MDM2, phospho Rb and total Rb protein level from nuclear extract of MCF7 (left) and T47D (right) cells are shown. Dot plot diagram shows quantified ImageJ values of phospho Rb protein levels normalized to lamin A from three independent experiments, when 1μl of purified MDM2 was added to the nuclear extracts of MCF7 (left) and T47D (right) cells. The p-value for MCF7 cells with overnight estrogen treatment and addition of purified MDM2 (compare lane 4 to lane 5) was statistically significant. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Western Blot, MTT Assay, Control, Plasmid Preparation, Activity Assay, Confocal Microscopy, Fluorescence, Staining, Phospho-proteomics, In Vitro, Kinase Assay, Purification

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Number of large colonies (50μm or larger) determined by counting the colonies of MCF7 cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA induction (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (160, 158, 175), control vector + shRNA induction (153, 151, 197), mdm2 shRNA –shRNA induction (140, 137, 200) and mdm2 shRNA + shRNA induction (17, 11, 8). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.002. (B) Number of large colonies (100μm or larger) determined by counting the colonies of T47D cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (220, 191, 70), control vector + shRNA induction (202, 195, 33), mdm2 shRNA –shRNA induction (166, 175, 69) and mdm2 shRNA + shRNA induction (27,20, 9). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.02. (C) Representative images of colonies that T47D cells formed in soft agar in the presence or absence of MDM2 knockdown. (D) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in presence or absence of 4μg/ml doxycycline, were fixed and stained with propidium iodide. Colony masses were categorized in 5 different groups and the number of masses in each group were counted and presented as percentages in the total population. This is an average of two independent experiments. The total number of masses scored for 2 independent experiments were control vector –shRNA induction (20, 15), control vector + shRNA induction (25, 27), mdm2 shRNA –shRNA induction (93, 95) and mdm2 shRNA + shRNA induction (86, 83). The p-value was determined by 2-tailed Student t-test. The p-value for large and small colonies for comparisons with and without MDM2 knockdown were p-value=0.03 and p-value=0.05 respectively. Two independent scorers counted the numbers of colonies for each independent experiment.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Fluorescence, Microscopy, Control, Plasmid Preparation, Knockdown, Staining

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in the presence or absence of 4 μg/ml dox, were fixed, stained with F-Actin and mounted with DAPI containing mounting media. Confocal z-stack images were acquired. Masses with lumen were counted and presented as percent of total number of masses grown in 3D matrigel. An average of two independent experiments are shown. The number of masses counted for 2 independent experiments were control vector -shRNA induction (21, 41), control vector +shRNA induction (21, 47), mdm2 shRNA -shRNA induction (31, 46) and mdm2 shRNA +shRNA induction (31, 62). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.01. Two independent scorers counted the numbers of masses for each independent experiment. (B) A representative image from confocal immunofluorescence microscopy showing a single slice from z-stack of DAPI, GFP and F-Actin of estrogen treated inducible clonal T47D.sh mdm2 cells grown in 3D-matrigel in the presence or absence of 4μg/ml doxycycline (dox) for 3 weeks. The top and middle rows show hollow lumen and ductal lumen respectively in the presence of shRNA expression to mdm2 ; the GFP (green) indicates shRNA induction to mdm2 . The third row shows mass structure (disruption of normal mammary glandular architecture) in the absence of shRNA expression to mdm2 .

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Staining, Control, Plasmid Preparation, shRNA, Knockdown, Immunofluorescence, Microscopy, Expressing, Disruption

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) & (B) T47D cells ((A) inducible sh mdm2 clonal and vector pool; (B) constitutive sh mdm2 and vector pool) grown in the presence of estrogen in 3D matrigel for 3.5 weeks. The cells were fixed, permeabilized, blocked, stained with phospho-histoneH3 antibody and mounted with DAPI containing mounting media. Images were taken with confocal microscope. Quantitative analysis of phospho-histone H3 positive cells were performed by capturing optical Z-stack sections of masses and dividing the number of positive phospho-histone H3 cells by the total number of masses. (A) The number of masses counted in each group for 2 independent experiments were control vector -shRNA induction (29, 30), control vector +shRNA induction (30, 30), mdm2 shRNA -shRNA induction (29, 30) and mdm2 shRNA +shRNA induction (30, 30). (B) The number of masses counted in each group for 2 independent experiments were control vector (49, 60) and sh mdm2 (48, 60). Average of two independent experiments are shown for each knockdown method. The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.001 (A) and p-value=0.009 (B). Two independent scorers counted the numbers of masses for each independent experiment. (C) Representative confocal Z-stack image (single slice) showing DAPI, phospho-histone H3 and GFP in estrogen-treated inducible clonal T47D.sh mdm2 cells in the presence and absence of 4μg/ml doxycycline. (D) Cell cycle analysis by FACS (Fluorescence Activated Cell Sorting). T47D cells were harvested, fixed and stained with propidium iodide and subjected to cell cycle analysis by FACS. Data are presented as percent of cells in S phase in a total population of 10,000 cells and analyzed by FACS in each group. Average of 4 independent experiments are shown. The p-value was determined by 2-tailed Student t-test and * represents a p-value ≤ 0.05

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Plasmid Preparation, Staining, Microscopy, Control, shRNA, Knockdown, Cell Cycle Assay, Fluorescence, FACS

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Inducible clonal MCF7 cells with mdm2 shRNA or control vector were treated with and without 2μg/ml doxycycline(dox) for 3 days to induce shRNA expression, followed by 10nM estrogen for 5 days in the presence and absence of dox. A representative image of western blot analysis of phospho Rb, Total Rb, E2F1, MDM2 and Actin protein levels from 50μg whole cell protein extract is shown. (B) ImageJ analysis was performed for phospho Rb, E2F1 and MDM2 protein levels normalized to Actin. Graph represents average of four independent experiments with standard deviation in inducible clonal of MCF7 cells with mdm2 shRNA or control vector. * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Control, Plasmid Preparation, Expressing, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: Model showing that MDM2 is a central hub in estrogen signaling and works through an Rb-E2F1 pathway to promote proliferation. Breast cancer cells harboring SNP309 have increased binding of the transcription factor Sp1, which causes elevated MDM2 protein levels. Fulvestrant blocks the MDM2 pathway and the Rb-E2F1 pathway.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay

Journal: The Febs Journal

Article Title: HAUSP, a novel deubiquitinase for Rb – MDM2 the critical regulator

doi: 10.1111/febs.12843

Figure Lengend Snippet: Mechanism of Rb regulation in glioma. (A) WB of Rb upon HAUSP overexpression in U87MG cells either with (right panel) or without (left panel) MG132 treatment (10 μ m ) for 14 h. (B) WB was performed to verify HAUSP knockdown and the effect on Rb protein expression using specific siRNA in U87MG cells. Actin serves as the loading control. (C) In vivo deubiquitination assay in U87MG cells in the presence of WT or CI HAUSP; IP was done with Rb and WB with Rb and Ub. (D) WB of Rb in H1299 (p53 null) cells upon HAUSP (WT or CI) overexpression with Ub cotransfected. (E) ICC for HAUSP and Rb in a panel of cancer cells with varied p53 background. H1299, p53 null (top); HCT116, p53 WT (middle); HT29, p53 mutant (lower). (F) WB of Rb in HEK293 cells cotransfected with HAUSP and MDM2. (H) Fluorimetric estimation of Rb expression in the presence of HAUSP alone or cotransfected with MDM2, represented as relative fold change with respect to GFP-Rb alone. P = 0.02 (*) and 0.007 (**). (G) Fluorescence emission spectra of cells transfected with either GFP-Rb alone (red graph, red line represents the mean value) or along with HAUSP (blue graph, blue line represents the mean value) or by increasing the load of MDM2 transiently (green graph, green line represents the mean value) keeping a negative control without GFP transfection (grey graph, grey line represents the mean value). (I) Graphical representation of cells distributed in the various phases of the cell cycle from FACS analysis (extended from the experiment shown in Fig. D,E,F). (J) WB of Rb upon HAUSP overexpression in U87MG cells with or without MDM2 inhibitor (10 μ m ) for 24 h (left panel) and in the absence or presence of siMDM2 (right panel). The values in (A), (F) and (J) have been estimated by densitometry and normalized against the loading control actin/GAPDH.

Article Snippet: Human cell lines GBM, U87MG and DBTRG-05MG; lung adenocarcinoma H1299 (p53 null); colon carcinoma HT29 (p53 mutated) and HCT116 (p53 WT); normal embryonic kidney HEK293; rat glioma cell line C6; and monkey normal kidney cell line COS-7 were procured from American Type Culture Collection (Manassas, VA, USA).

Techniques: Over Expression, Knockdown, Expressing, Control, In Vivo, Mutagenesis, Fluorescence, Transfection, Negative Control