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: 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: 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: Journal of Biological Chemistry

Article Title: Activation of Targeted Necrosis by a p53 Peptide

doi: 10.1074/jbc.m701864200

Figure Lengend Snippet: FIGURE 1. p53p-Ant uptake. A, DU-145 or PC-3 cells exposed to 30 M p53p-Ant for up to 10 min. Anti-p53 pAb421 antibody (epitope 371–380 aa) detects peptide. The blots were stripped and reprobed with anti-p53 DO-1 antibody (epitope 11–25 aa) to confirm endogenous p53 status. B, DU-145 cells were untreated (CONT) or incubated with 30 M p53p or p53p-Ant for 10 min. The cell lysates were analyzed as above. C, DU-145 cells incubatedwith30Mp53p-Ant-RhoBandfluorescencemonitoredbyconfocalmicroscopy.Thesamegroupof cells was followed for up to 10 min. Untreated cells showed no staining. Low intensity fluorescence is repre- sented by gray (background color), medium intensity is white, and high intensity is black.

Article Snippet: The human androgen-independent prostate cancer cell lines PC-3 (null p53), DU-145 (mutant p53, Leu-223 and Phe-274), 22Rv1 (mutant p53, Arg-331), VCaP (mutant p53, Trp-248), and LNCaP (wt p53) were obtained from ATCC (Manassas, VA).

Techniques: Incubation, Staining, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Activation of Targeted Necrosis by a p53 Peptide

doi: 10.1074/jbc.m701864200

Figure Lengend Snippet: FIGURE 2. Morphological changes and growth inhibition induced by p53p-Ant treatment. A, DU-145 or PC-3 cells were untreated (CONT) or exposed to 30 M p53p-Ant. The cells were photographed under phase con- trast microscopy for up to 6 h. B and C, DU-145 (B) or PC-3 (C) cells were untreated (CONT) or exposed to 30 M Ant, p53p, or p53p-Ant for 3 to 24 h or to 10, 20, or 30 M p53p-Ant for 6 h. For B and C, cell viability was determined byMTTassay.TheresultsrepresentmeanabsorbanceS.D.(n3).D,trypan blue cell death in PC-3 cells or PC-3 stably transfected cells expressing a tem- perature-sensitive p53-143 at 37 °C (mutant p53) or 32 °C (wt p53) for 3 h. At least 200 cells were counted in triplicate.

Article Snippet: The human androgen-independent prostate cancer cell lines PC-3 (null p53), DU-145 (mutant p53, Leu-223 and Phe-274), 22Rv1 (mutant p53, Arg-331), VCaP (mutant p53, Trp-248), and LNCaP (wt p53) were obtained from ATCC (Manassas, VA).

Techniques: Inhibition, Microscopy, Stable Transfection, Transfection, Expressing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Activation of Targeted Necrosis by a p53 Peptide

doi: 10.1074/jbc.m701864200

Figure Lengend Snippet: FIGURE 9. Pathways of nonspecific necrosis, targeted necrosis, and apoptosis. Wild type p53, through the action of ROS, Fas, or Bcl-2 family of proteins, activates caspases, resulting in apoptosis. Necrosis is activated by injury or perfusion defects, leading to generation of ROS. Alternatively, p53p-Ant preferentially binds to mutant p53 and alters its conformation resulting in functional p53. The functional mutant p53 may mediate gen- eration of ROS leading to DNA nicks, nucleosomal degradation, and mito- chondrial membrane damage with loss of ATP pools but without caspase-3 activation. This leads to targeted necrosis because it will only be activated in mutant p53 cancer cells, which express sufficient threshold p53 target for the peptide and decreased ATP, which commits the cell to undergo necrosis. Alternatively, functional p53 may act via the Fas/FADD pathway leading to apoptosis, as in breast cancer cells, which do not undergo massive loss of ATP.

Article Snippet: The human androgen-independent prostate cancer cell lines PC-3 (null p53), DU-145 (mutant p53, Leu-223 and Phe-274), 22Rv1 (mutant p53, Arg-331), VCaP (mutant p53, Trp-248), and LNCaP (wt p53) were obtained from ATCC (Manassas, VA).

Techniques: Mutagenesis, Functional Assay, Membrane, Activation Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Differential enhancement of a cutaneous HPV promoter by DeltaNP63alpha, Jun and mutant p53.

doi: 10.4161/cc.4.5.1653

Figure Lengend Snippet: Figure 2. (A) Deletion constructs of the pGL3- 20URR-En were co-transfected with ∆Np63α, with and without subsequent UV-irradiation of the cells, and luciferase assays performed. The deletion of the region between nt128 and nt195 led to a significant decrease in the activation by ∆Np63α. The pGL3-20URR-En reporter construct alone was used as control. (B) The HPV 20 enhancer (nt142- nt178) does not bind to ∆Np63α. The radiola- belled oligonucleotide 20-1, differing in four nucleotides from a p53 canonical binding site, was used in electrophoretic mobility assays with protein extracts either from H1299 cells alone, or from H1299 cells transfected with wild type p53. Radiolabelled Gadd-45 oligonucleotide was used as control. Supershift was observed with the Gadd-45 oligonucleotide in the presence of p63 antibody (H129), as well as p53 antibody. Protein/DNA complex was not observed using the radiolabelled 20-1 oligonucleotide.

Article Snippet: The following primary antibodies were used: monoclonal DO-1 antibody (1:1000 dilution, Santa Cruz SC-126) for detection of wild type and mutant p53 proteins, anti-p63α antibody H-129 (200 μg/0.1 ml, 1:2000 dilution, Santa Cruz sc-8344X), anti-JunD 329X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc-74X), anti-JunB N-17X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc-46X), anti-c-Jun H-79 (200 μg/ml, 1:200 dilution, Santa Cruz sc-1694) and anti-p-c-Jun KM-1X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc- 8344X).

Techniques: Construct, Transfection, Irradiation, Luciferase, Activation Assay, Control, Binding Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Differential enhancement of a cutaneous HPV promoter by DeltaNP63alpha, Jun and mutant p53.

doi: 10.4161/cc.4.5.1653

Figure Lengend Snippet: Figure 4. Co-transfection of the HPV 20-URR-luciferase reporter construct, pGL3-20URR with c-jun, ∆Np63α and p53 mutant R248W into H1299 cells. (A) The activation measured by co-expression of ∆Np63α was further increased in the presence of co-expressed c-Jun. The activation of the viral promoter in H1299 co-transfected with ∆Np63α and c-Jun, was downregu- lated in the presence of co-transfected mutant R248W. (B) Western blot analyses of the transfected cells used in (A). The co-expression of mutant R248W protein with c-Jun and ∆Np63α leads to a decrease in the protein levels of the ∆Np63α and c-Jun. (C) The level of c-jun transcription remains constant independent of the overexpression of ∆Np63α. mRNA was extracted from transfected cells and reverse transcriptase PCR amplification performed. RT-PCR amplification of GAPDH was used as control.

Article Snippet: The following primary antibodies were used: monoclonal DO-1 antibody (1:1000 dilution, Santa Cruz SC-126) for detection of wild type and mutant p53 proteins, anti-p63α antibody H-129 (200 μg/0.1 ml, 1:2000 dilution, Santa Cruz sc-8344X), anti-JunD 329X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc-74X), anti-JunB N-17X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc-46X), anti-c-Jun H-79 (200 μg/ml, 1:200 dilution, Santa Cruz sc-1694) and anti-p-c-Jun KM-1X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc- 8344X).

Techniques: Cotransfection, Luciferase, Construct, Mutagenesis, Activation Assay, Expressing, Transfection, Western Blot, Over Expression, Reverse Transcription, Amplification, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Differential enhancement of a cutaneous HPV promoter by DeltaNP63alpha, Jun and mutant p53.

doi: 10.4161/cc.4.5.1653

Figure Lengend Snippet: Figure 7. (A) Luciferase reporter assays demonstrate that the co-expression of c-Jun increases the activation of the viral promoter by ∆Np63α, whereas wtp53 downregu- lates this activation by ∆Np63α. (B) Co-immunoprecipitation assays using antibodies directed against either p-c-Jun or p53 (DO-1), resulted in co-precipitation of ∆Np63α, p-c-Jun as well as wtp53, as shown in western blot analyses of these complexes. ∆Np63α and p-c-Jun were not immunoprecipitated with β-actin antibody.

Article Snippet: The following primary antibodies were used: monoclonal DO-1 antibody (1:1000 dilution, Santa Cruz SC-126) for detection of wild type and mutant p53 proteins, anti-p63α antibody H-129 (200 μg/0.1 ml, 1:2000 dilution, Santa Cruz sc-8344X), anti-JunD 329X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc-74X), anti-JunB N-17X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc-46X), anti-c-Jun H-79 (200 μg/ml, 1:200 dilution, Santa Cruz sc-1694) and anti-p-c-Jun KM-1X (200 μg/0.1 ml, 1:5000 dilution, Santa Cruz sc- 8344X).

Techniques: Luciferase, Expressing, Activation Assay, Immunoprecipitation, Western Blot