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: 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

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: Expression levels of DPEP2 in generalized carcinoma and LUAD. (A) The heatmap of the top ten upregulated and downregulated genes with LUAD progression based on RNA sequencing data in Gene Expression Omnibus (GSE31210). (B) The expression level of DPEP2 in different tumors in TCGA was determined using TIMER2.0. (C) The cBioPortal OncoPrint map shows the distribution of DPEP2 genome changes in patients with LUAD. (D) Expression level of DPEP2 in normal tissues and tumor tissues. (E) Expression level of DPEP2 in normal tissues and the paired adjacent tumor tissues. (F) Transcriptional level of DPEP2 in the human lung bronchial epithelial cell line BEAS-2B and various LUAD cell lines (A549, H1650, and H1299). (G) Validation of the expression level of DPEP2 in LUAD using the Human Protein Atlas database (immunofluorescence). (H) The protein levels of DPEP2 were determined using western blotting in different LUAD cell lines. *P<0.05, **P<0.01 and ***P<0.001; two-tailed Student's t-test. DPEP2, dipeptidase-2; LUAD, lung adenocarcinoma.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: Expressing, RNA Sequencing, Gene Expression, Biomarker Discovery, Immunofluorescence, Western Blot, Two Tailed Test

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: DPEP2 affects migration, invasion, and EMT of lung adenocarcinoma cells. (A) Expression of DPEP2 in empty vector control (Vector) and DPEP2-overexpressing cells (DPEP2) was detected using western blotting and reverse transcription-quantitative PCR assays. GAPDH served as a loading control. (B) Representative images and quantitative analysis of wound-healing assay of A549 and H1650 cells (scale bar, 200 µm). (C and D) Transwell representative images and analysis of (C) cell migration and (D) invasion (scale bar, 100 µm). (E and F) The EMT markers (E-cadherin, N-cadherin, vimentin, and α-SMA) were analyzed using western blot analysis and IF staining (scale bar, 50 µm). Histograms represent the mean ± standard deviation based on three independent experiments. *P<0.05, **P<0.01 and ***P<0.001, two-tailed Student's t-test. DPEP2, dipeptidase-2; EMT, epithelial-mesenchymal transition.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: Migration, Expressing, Plasmid Preparation, Control, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Wound Healing Assay, Staining, Standard Deviation, Two Tailed Test

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: DPEP2 enhances lung adenocarcinoma cell sensitivity to cisplatin by regulating cancer stem cell transformation. (A and B) Expression of CD44 and CD133 was determined by western blotting and immunofluorescence staining (scale bar, 50 µm). (C) Representative images and analysis of sphere formation in A549 and H1650 cells (scale bar, 100 µm). (D) Representative images of colony formation assays of A549 and H1650 cells treated with various concentrations of cisplatin at 0, 2, 4 or 8 µg/ml. (E and F) Cell Counting Kit-8 assay was used to analyze the sensitivity of A549 and H1650 cells to various concentrations of cisplatin at 0, 2, 4 or 8 µg/ml. **P<0.01, two-way ANOVA with post hoc Dunnett's t-test. (G) Representative flow cytometric images of A549 and H1650 cells treated with 0 or 2 µg/ml cisplatin. **P<0.01 and ***P< 0.001, two-tailed Student's t-test. DPEP2, dipeptidase-2.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: Transformation Assay, Expressing, Western Blot, Immunofluorescence, Staining, Cell Counting, Two Tailed Test

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: DPEP2 enhances the sensitivity of lung adenocarcinoma cells to cisplatin in vivo . (A) Flow chart of the in vivo experiment with nude mice. (B and C) Nude mice were subcutaneously injected with a vector or DPEP2-overexpressing A549 stable strain. On days 7 and 14, mice were injected with PBS or cisplatin (20 mg/m 2 ). Every 7 days, the tumor volume was measured with calipers. Tumor volume on day 28 was assessed using a two-tailed Student's t-test (vector vs. DPEP2; and vector + cisplatin vs. DPEP2 + cisplatin). *P<0.05 and ***P<0.001. (D and E) On day 28, tumors were excised and weighed. Representative tumors isolated from nude mice and average tumor weights. **P<0.01 and ***P<0.001, one-way ANOVA with post hoc Dunnett's t-test. (F and G) Immunohistochemical staining of DPEP2, epithelial-mesenchymal transition-associated genes (E-cadherin, N-cadherin, vimentin, and α-SMA), stem cell biomarkers (CD44 and CD133), and the proliferation marker Ki67 in tumors of mice injected with vector or DPEP2-overexpressing cells (scale bar, 100 µm). DPEP2, dipeptidase-2.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: In Vivo, Injection, Plasmid Preparation, Two Tailed Test, Isolation, Immunohistochemical staining, Staining, Marker

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 1. tp53 suppresses embryonal rhabdomyosarcoma (ERMS) tumor initiation. (A) Kaplan–Meier plot showing ERMS tumor initiation in tp53-/- and tp53+/+ fish. (B) Representative images of DsRed-positive zebrafish ERMS. Arrows show tumor location for each fish. All tumor-burdened zebrafish are 10 days old. Scale bar = 0.5 mm. (C) Tumor numbers per zebrafish in tp53-/- and tp53+/+ fish. n = 44 (tp53+/+), n=130 (tp53-/-). (D) Ratio of tumor area to total body area in in tp53-/- and tp53+/+ fish. n = 10. (E) Pie chart showing percentage of tumors found in varying regions of tp53-/- and tp53+/+ fish, showing no significant differences in tumor localization. Head – p=0.25848, trunk – p=0.39532, tail – p=0.92034 (two-tailed two proportions Z-test). (F) Representative H&E staining of zebrafish ERMS tumors. Scale bar = 100 µm.

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Two Tailed Test, Staining

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 2. tp53 is a potent suppressor of proliferation and to a lesser extent of apoptosis. (A) Representative confocal microscopy images of EdU staining on embryonal rhabdomyosarcoma (ERMS) tumor sections and a plot quantifying the percentage of EdU-positive cells. Average of n = 8–11/primary tumors. White arrows show EdU-positive cells. (B) Representative confocal microscopy images of phospho-histone H3 staining on ERMS tumor sections (scale bar = 100 µm). Total number pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from n7-9 primary tumors. One the right-most panel is a plot quantifying the total number of pHH3-positive cells per single ERMS section. White arrows show pHH3-positive cells. (C, D) Representative flow cytometry analysis of Annexin V staining of tp53+/+and tp53-/- ERMS tumors, respectively. (E) Quantification of flow cytometry analysis of Annexin V staining. Q1 = pre-necrotic cells, Q2 = late apoptosis + necrotic cells, Q3 = living cells, Q4 = early apoptotic cells. n = 7. ns, not significant, p=0.5926, unpaired t-test.

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Confocal Microscopy, Staining, Flow Cytometry

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 3. Human TP53 blocks tumor initiation, growth, and proliferation and increases apoptosis in tp53-/- zebrafish. (A) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53-/- fish with or without p53WT expression. Western blot analysis was performed to assess p53WT expression level in tumors. (B) Representative images of ERMS tumors in tp53-/- fish with or without human TP53WT expression. Tumor-burdened zebrafish are between 15 and 20 days old. Scale bar = 1 mm. (C) Ratio of tumor area to total body area in tp53-/- fish with or without expression of TP53WT. n = 18. (D) Number of tumors per tp53-/- zebrafish with or without expression of TP53WT. ns, not significant. n = 36 (tp53-/-), n = 28 (TP53WT). (E) Pie chart showing site of tumor localization in tp53-/- fish with or without expression of TP53WT showing no statistical differences. Head – p=0.20045, trunk – p=0.42858, tail – p=0.3336. Quantification of proliferation (F) and apoptosis (G) via EdU staining (n = 10) and Annexin V staining (n = 3), respectively, for tumors arising in tp53-/- fish with or without expression of TP53WT.

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Expressing, Western Blot, Staining

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 4. Assigning pathogenicity to two human TP53 sarcoma mutations in the kRASG12D-driven embryonal rhabdomyosarcoma (ERMS) model. (A) Lollipop plot showing the two novel, human p53 mutations P153Δ and C176F, as well as the amino acid sequence alignment for human, mouse, and zebrafish protein. (B) DNA sequencing data from osteosarcoma patient confirming the germline P153Δ mutation, as well as somatic A159V mutation. (C, D) p53 immunohistochemistry staining of p53 in ERMS patient-derived xenograft (PDX) SJRHB00011 expressing p53C176F and osteosarcoma expressing p53P153Δ. (E, F) Representative H&E staining of ERMS PDX expressing the C176F mutation and diagnostic biopsy of osteosarcoma tumor expressing osteosarcoma expressing p53P153Δ showing neoplastic tumor cells with pleomorphic nuclei, irregular chromatin pattern, as well as irregular disorganized trabeculae of unmineralized malignant osteoid (stars). (G) Protein expression of mutant p53 in zebrafish ERMS tumors, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. (H) Kaplan–Meier plot showing tumor initiation in tp53-/- fish with or without expression of mutant TP53. (I) Representative images of tumor localization in tp53-/- fish with or without expression of mutant TP53. Age of zebrafish in panels is 37 d. Scale bar = 1 mm (J) Pie chart showing percentage of tumors found in varying regions of tp53-/- fish with or without expression of mutant TP53. Percentages in red indicate a significant difference to tp53-/- (p=0.0096, two-tailed two proportions Z-test). (K) Quantification of Annexin V staining in tumors arising in tp53-/- fish with or without

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Sequencing, DNA Sequencing, Mutagenesis, Immunohistochemistry, Staining, Derivative Assay, Expressing, Diagnostic Assay, Control, Two Tailed Test

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 5. Expression of TP53P153Δ with kRASG12D in tp53-/- zebrafish results in the initiation of medulloblastomas with a shh gene signature. (A) Heatmap from RNAseq analyses comparing tumors expressing kRASG12D;tp53-/- to kRASG12D; tp53-/-; TP53P153Δ (n = 3/group). A total of 643 genes were selected for the heatmap, with adjusted p-value<0.01 and fold-change >10. (B) Enriched Gene Ontology (GO) Biological Processes (BP) in upregulated genes in p53-/- group (left panel with pink bars) consistent with the expected tissue of origin for kRASG12D;tp53-/- to kRASG12D; tp53-/-; TP53P153Δ (right panel

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Expressing

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 6. TP53Y220C predisposes to head embryonal rhabdomyosarcoma (ERMS) in zebrafish. (A) Surface representation of p53WT (PDB 2XWR) and p53P153Δ (homology model) showing key residues lining a surface exposed pocket (sticks). The green ovals compare the size and shape of the pocket between the two structures. (B) p53 protein expression levels in tp53-/- fish tumors with or without TP53Y220C, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. (C) Kaplan–Meier plot showing tumor initiation in tp53-/- fish, with or without TP53Y220C. (D, F) Representative images of tp53-/- fish with ERMS tumors, with or without TP53Y220C (GFP-positive). Dashed region outlines the tumor. The zebrafish in (F) are 35 d. Scale bar in (F) 1 mm. (E, G) Representative H&E staining of tumors in tp53-/- fish, with or without TP53Y220C. Scale bar = 100 µm. (H) Pie chart showing localization of tumors expressed as a percentage found in varying regions of in tp53-/- fish with and without TP53Y220C. Percentage in red indicates a significant difference to tp53-/- (p=0.01928, two-tailed two proportions Z-test). (I) Quantification of Annexin V staining in tumors of tp53-/- fish with or without expression of TP53Y220C. n = 3–4. (J) Quantification of EdU staining in tumors of tp53-/- fish with or without expression of TP53Y220C. n = 4–9.

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Expressing, Control, Staining, Two Tailed Test

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 7. kdr downstream of TP53P153Δ predisposes to head embryonal rhabdomyosarcoma (ERMS) in tp53-/- zebrafish. (A) Volcano plot comparing head to trunk ERMS tumors. Genes with adj. p-value<0.01 and fold-change >2 are colored in red (upregulated in head tumor) or blue (downregulated in head tumor). Table adjacent the plot shows the top differentially expressed genes. (B) Western blot showing p53, KDR, and RUNX2 protein expression in bone marrow mesenchymal cells, the osteosarcoma PDX expressing TP53P153Δ, and osteosarcoma cell lines SaOS2 and 143B. (C) Representative KDR

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma.

doi: 10.1038/sj.onc.1208755

Figure Lengend Snippet: Figure 2 Cisplatin induction of selected genes in NT2/D1 cells is dependent on p53. (a) p53 siRNA efficiently suppresses basal p53 mRNA expression and cisplatin induction of p21 mRNA and p53 protein. NT2/D1 cells were transfected with no siRNA, scrambled control siRNA (Scb), and two p53 siRNAs and treated with 0, 0.5, and 2.0 mM cisplatin for 6 h. Adherent cells were harvest 24 h later for Northern and Western analysis. Representative of three independent experiments. (b) p53 siRNA blocks cisplatin induction of p53 transcriptional activity. MDM2-based reporter assay was performed in NT2/D1 cells cotransfected with insertless vector, a dominant- negative p53 construct (DN p53), scrambled siRNA, or two different p53 siRNAs as described in Materials and methods. Error bars are s.d. Representative of three independent experiments. (c) Effect of cisplatin and p53 siRNA on transcript levels of selected genes from Table 1. Cells were treated as in Figure 2a and RT–PCR analysis was performed for the indicated genes. Fold-induction compared to untreated wild-type cells as determined by densitometry is indicated below each band. In indicated samples (RT), reverse transcriptase was omitted to control for genomic contamination. Representative of two independent experiments. (*) Indicates genes known from prior studies to be p53 targets

Article Snippet: The p53 antibody (DO-1) and the FAS antibody were from Santa Cruz.

Techniques: Expressing, Transfection, Control, Northern Blot, Western Blot, Activity Assay, Reporter Assay, Plasmid Preparation, Dominant Negative Mutation, Construct, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Oncogene

Article Title: A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma.

doi: 10.1038/sj.onc.1208755

Figure Lengend Snippet: Figure 3 Dose response of array gene induction following cytotoxic treatments. (a) NT2/D1 cells were treated with the indicated dosages of cisplatin and etoposide for 6 h and drug was then removed for 24 h before harvest. Cells were treated with anisomycin continuously for 4 h. Expression of PLK2, GDF15, FLJ11259, FAS, and p21 were assessed by RT–PCR on adherent cells. Stabilization of p53 and activation of caspase 3 were assessed via Western analysis on adherent and floating cells with p53 antibody (Santa Cruz) and an antibody that specifically recognizes activated caspase 3 (Cell Signaling). Array genes are induced with lethal and supralethal doses of cisplatin and etoposide but not supralethal doses of anisomycin. Ansiomycin activates caspase 3 independent of p53 activation. (b) Transplatin does not induce array genes. NT2/D1 cells were treated with the indicated doses of cisplatin or transplatin for 6 h and assayed 24 h later for PLK2, GDF15, FLJ11259, FAS, and p21 expression by RT–PCR and p53 via Western analysis. Doses of transplatin as high as 10 mM failed to induce p53 or cisplatin-induced genes

Article Snippet: The p53 antibody (DO-1) and the FAS antibody were from Santa Cruz.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Western Blot

Journal: Oncogene

Article Title: A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma.

doi: 10.1038/sj.onc.1208755

Figure Lengend Snippet: Figure 4 Cisplatin target genes are induced at the protein level despite inhibition of apoptosis. (a) Western analysis depicting cisplatin induction of FDXR and FAS. Induction was blunted in cells treated with p53 siRNA. NT2/D1 cells were transfected with scrambled control siRNA (Scb) and p53 siRNA and treated with the indicated dosages of cisplatin for 6 h. Adherent cells were harvest 24 h later for Western analysis. (b) Cisplatin induction of FDXR and FAS occurs in the presence of a caspase 3 inhibitor. NT2/D1 cells were pretreated with 50 mM of the caspase-3 inhibitor Z-VAD-FMK for 1 h prior to addition of the indicated dosages of cisplatin. Adherent and floating cells were harvested 24 h later for Western analysis. Inhibition of apoptosis was assessed by abroga- tion of PARP cleavage

Article Snippet: The p53 antibody (DO-1) and the FAS antibody were from Santa Cruz.

Techniques: Inhibition, Western Blot, Transfection, Control

Journal: Oncogene

Article Title: A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma.

doi: 10.1038/sj.onc.1208755

Figure Lengend Snippet: Figure 5 Scatter plot analysis comparing expression-hybridiza- tions from cisplatin-treated, wild-type and p53 siRNA cells. Individual genes were plotted using the log of the normalized signal intensity for each hybridization. Cisplatin treatments were 0.5 mM for 6 and 24 h recovery. (a) Comparison of gene expression in untreated p53 siRNA cells (y-axis) and untreated wild-type NT2/ D1 cells (x-axis). Genes below the diagonal are repressed in p53 siRNA cells. Two independent probe sets for p53 are indicated. (b) Comparison of gene expression in cisplatin-treated, wild-type NT2/ D1 cells (y-axis) and untreated wild-type NT2/D1 cells (x-axis). Genes above the diagonal are induced with cisplatin in NT2/D1 cells. This is a representation of the data in Table 1. (c) Comparison of gene expression in cisplatin-treated p53 siRNA cells (y-axis) and untreated p53 siRNA cells (x-axis) indicates extensive disruption of cisplatin-mediated target gene regulation following p53 knockdown in NT2/D1 cells

Article Snippet: The p53 antibody (DO-1) and the FAS antibody were from Santa Cruz.

Techniques: Expressing, Hybridization, Comparison, Gene Expression, Disruption, Knockdown

Journal: Oncogene

Article Title: A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma.

doi: 10.1038/sj.onc.1208755

Figure Lengend Snippet: Figure 6 p53-dependent cisplatin response in NT2/-D1 cells. (a) Survival assays were performed in NT2/D1 cells untransfected (no siRNA) or transfected with control siRNA (Scb) or p53 siRNA. Transfected cells were plated at a density of 5 104 cells per well of a six-well plate and the next day exposed to indicated dosages of cisplatin for 6 h. Cell numbers were determined 4 days later by hemocytometer count of viable Trypan blue excluded cells. Data are normalized to viable cell number in the absence of cisplatin (control). Average of triplicate treatments. Error bars are standard error of the mean (s.e.m.). Representative of three independent experiments. (b) Scramble siRNA (Scb) or p53 siRNA cells were untreated or treated with 10 mM cisplatin or 10 mg/ml anisomycin and analysed for loss of mitochondrial membrane potential using DiOC6 staining and FACS analysis as described in Material and methods. Percentages indicate number of cells with membrane potential loss. Cisplatin treatment was for 6 h followed by 24 h incubation in drug-free media. Anisomycin treatment was for 4 h. Adherent and floating cells were assessed in all cases

Article Snippet: The p53 antibody (DO-1) and the FAS antibody were from Santa Cruz.

Techniques: Transfection, Control, Membrane, Staining, Incubation

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

doi: 10.1074/jbc.m407450200

Figure Lengend Snippet: FIG. 1. WT p53 inhibits PKC protein expression in human STS cells. A, 200 g of total cell lysates obtained from SKLMS-1 (mut p53), U2-OS (WT p53), Saos-2 (p53 null), and HT1080 (p53-deficient) cells were analyzed by Western blotting with an anti-PKC antibody. -Actin was used as a loading control, and p21 was used as WT p53 function marker. B, protein obtained from SKLMS-1, SKNeo, SKAla-1, SKAla-2, and SKAla-3 cells was analyzed by Western blotting. SKAla cells are SKLMS-1 cells transfected with Ala-143 temperature-sensitive p53 mutant expressing vector and cultured at 32 °C with WT p53 or 38 °C with mut p53, respectively. C, Saos-2 cells were transiently transfected with an Ala-143 temperature-sensitive mut p53 expressing vector and cultured at 32 and 38 °C, respectively, as described under “Experimental Proce- dures.” Cell lysates were prepared, and Western blotting was performed as described in A.

Article Snippet: Cell Lines—The cell lines SKLMS-1 (a human leiomyosarcoma cell line harboring a p53 point mutation at codon 245) (24) and HT1080 (a fibrosarcoma cell line deficient in p53) (25) were obtained from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium with 10% fetal bovine serum (complete culture medium; Invitrogen) in 5% CO2 at 37 °C.

Techniques: Expressing, Western Blot, Control, Marker, Transfection, Mutagenesis, Plasmid Preparation, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

doi: 10.1074/jbc.m407450200

Figure Lengend Snippet: FIG. 2. Wt p53 inhibits PKC mRNA expression in human STS cells. A, 30 g of total RNA from SKLMS-1, SKNeo, SKAla-1, SKAla-2, and SKAla-3 cells was analyzed by Northern blotting. Northern blotting analysis of GAPDH was used as a loading control. B, 30 g of total RNA from Saos-2 cells, which were transfected with Ala-143 temperature-sensitive mu- tant p53-expressing vectors as described in Fig. 1C, was analyzed by Northern blotting. C, temperature-sensitive p53 mutant SKAla-2 cells were cultured at 32 or 38 °C for 72 h. The cells were then shifted from 32 to 38 °C or from 38 to 32 °C, and total RNA was prepared on days 0, 2, 4, and 6 and analyzed by North- ern blotting for PKC expression.

Article Snippet: Cell Lines—The cell lines SKLMS-1 (a human leiomyosarcoma cell line harboring a p53 point mutation at codon 245) (24) and HT1080 (a fibrosarcoma cell line deficient in p53) (25) were obtained from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium with 10% fetal bovine serum (complete culture medium; Invitrogen) in 5% CO2 at 37 °C.

Techniques: Expressing, Northern Blot, Control, Transfection, Mutagenesis, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

doi: 10.1074/jbc.m407450200

Figure Lengend Snippet: FIG. 3. Wt p53 Inhibits PKC mRNA expression through transcriptional repression not affecting PKC mRNA stability. A, PKC mRNA expression was analyzed by nuclear runoff assay. Nuclei from SKLMS-1 and SKAla-2 cells that were cultured at 32 and 38 °C, respectively, were collected, and transcription rates were determined by nuclear runoff assay as described under “Experimental Procedures.” PKC signals were normalized to respective GAPDH signals, and the PKC mRNA inhibition rate was calculated by comparing the PKC signal at 32 °C with WT p53 with that at 38 °C with mut p53. Data are representative of at least three independent experiments. B, PKC mRNA half-life was not affected by p53 statues. SKLMS-1 and SKAla-2 cells cultured at 32 and 38 °C, respectively, were treated with the transcription inhibitor actinomycin D (5 g/ml). Total RNA was isolated at various time points (0, 12, and 24 h) and analyzed by Northern blotting. The PKC signal was normalized to the basal level (at 0 h), and the half-life of the PKC mRNA was determined with the regression curves. Mean S.E. values from three independent experiments.

Article Snippet: Cell Lines—The cell lines SKLMS-1 (a human leiomyosarcoma cell line harboring a p53 point mutation at codon 245) (24) and HT1080 (a fibrosarcoma cell line deficient in p53) (25) were obtained from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium with 10% fetal bovine serum (complete culture medium; Invitrogen) in 5% CO2 at 37 °C.

Techniques: Expressing, Cell Culture, Inhibition, Isolation, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

doi: 10.1074/jbc.m407450200

Figure Lengend Snippet: FIG. 4. Localization of the PKC promoter region responsible for WT p53-mediated suppression of PKC transcription. A, PKC promoter deletion constructs were generated by polymerase chain reaction followed by cloning as described under “Experimental Procedures”. B, dose-dependent inhibition of PKC promoter-luciferase activity by Ad-p53. SKLMS-1 cells were pretreated with various doses of Ad-p53 or Ad-LacZ for 48 h. The cells were then cotransfected with 1 g of the PKC promoter-luciferase construct (1571/77) with 0.5 g of pSV40--galactosidase. Twenty-four hours later the luciferase activity was measured as described under “Experimental Procedures.” C, PKC promoter deletion constructs were transiently transfected into SKLMS-1 cells that were pretreated with Ad-p53 or Ad-LacZ (1000 virus particles/cell), and luciferase activities were determined on cells extracts and normalized to -galactosidase activity. Values are expressed as the percentage of the relative luciferase activity (100%) in cell extracts from untreated SKLMS-1 cells transfected with PKC promoter constructs. Mean S.E. of duplicate of three independent assays. *, p 0.05. D, Sp1 binding site of the PKC promoter sequence responsible for p53-mediated inhibition. E, relative luciferase activity was measured in wild type 260/77 and mutant 260/77 PKC constructs as in C. F, identification of specific Sp1 binding to the 34-bp PKC promoter region by electrophoretic mobility shift assay. Nuclear extracts from SKLMS-1 cells were incubated with radiolabeled mutant Sp1 oligonucleotides (lane 1) or wild type Sp1 oligonucleotides alone (lane 2) or plus anti-Sp1 antibody (lane 3). SKLMS-1 nuclear extracts were also preincubated with 100 unlabeled wild type Sp1 oligonucleotides and then were incubated with radiolabeled wild type Sp1 oligonu- cleotides (lane 4). The reactions were analyzed on 4% polyacrylamide gels containing 0.25 Tris borate/EDTA buffer. Ab, antibody.

Article Snippet: Cell Lines—The cell lines SKLMS-1 (a human leiomyosarcoma cell line harboring a p53 point mutation at codon 245) (24) and HT1080 (a fibrosarcoma cell line deficient in p53) (25) were obtained from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium with 10% fetal bovine serum (complete culture medium; Invitrogen) in 5% CO2 at 37 °C.

Techniques: Construct, Generated, Polymerase Chain Reaction, Cloning, Inhibition, Luciferase, Activity Assay, Transfection, Virus, Binding Assay, Sequencing, Mutagenesis, Electrophoretic Mobility Shift Assay, Incubation

Journal: Nature Communications

Article Title: Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer

doi: 10.1038/s41467-024-47080-3

Figure Lengend Snippet: a Kaplan-Meier plot of the correlation between the mutation of p53 and the survival of patients with NSCLC (Log-rank Mantel–Cox test). Cisplatin (Cis), fluvastatin (Flu). b Outline of the assays of cisplatin treatment (low dose, 10 μM; moderate (mod) dose, 25 μM; high dose, 50 μM). Cholesterol (Cho). Heatmap of the number of mutations and RNA-seq analysis ( c ), ROS levels ( d ), Cho levels ( e ), and DNA damage ( f ) for wtp53-expressing cells treated with cisplatin for 30 days. g High-throughput sequencing before and after fluvastatin sodium (4 μM) treatment in A549 cells. h Genome-wide analysis. The volcano plot depicts the significance and magnitude of difference (Fold Change). The dashed line indicates the threshold of the Fold Change > 2 and adjusted p < 0.05. Some of the cancer related genes are labeled by dark colors. i GO enrichment analysis of differentially expressed genes (DEGs). The advanced bubble chart shows GO enrichment of DEGs in signaling pathways. The x-axis label represents the gene ratio, and the y-axis label represents GO terms. j GSEA analysis. The normalized enrichment scores (NES) and p values are indicated in each plot. k Heatmap analysis of mevalonate pathway genes from RNA-seq data. The color scale indicates the fold change in genes expression. l Schematic illustration of treatment with cisplatin. b , g created with BioRender.com. Data are shown as the mean ± SD; n.s. = no significance. Source data are provided as a file.

Article Snippet: The p53 mutant human NSCLC cell lines NCI-H1975 (American Type Culture Collection (ATCC), #CRL-5908), NCI-H2087 (ATCC, #CRL-5922), NCI-H2342 (ATCC, #CRL-5941), NCI-H1793 (ATCC, #CRL-5896), NCI-H2196 (ATCC, #CRL-5932), NCI-H748 (ATCC, #CRL-5841), NCI-H1770 (ATCC, #CRL-5893), NCI-H1591 (ATCC, #CRL-5878), and NCI-H520 (ATCC, #HTB-182) were purchased from ATCC.

Techniques: Mutagenesis, RNA Sequencing, Expressing, Next-Generation Sequencing, Genome Wide, Labeling, Protein-Protein interactions

Journal: Nature Communications

Article Title: Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer

doi: 10.1038/s41467-024-47080-3

Figure Lengend Snippet: a Schematic summary of the p53-independent antitumor mechanism of FP NPs. b Confocal images of Dil and ER-Tracker in H1975 cells treated with 2 μM Dil@FP NPs. Their colocalization determined by Pearson’s correlation coefficient was quantified ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. c Confocal images of Dil and Fluo-4 in H1975 cells treated with 2 μM Dil@FP NPs. Their fluorescence intensity was quantified ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. d Confocal images of Dil and MitoTracker in H1975 cells treated with 2 μM Dil@FP NPs, and their colocalization determined by Pearson’s correlation coefficient was quantified ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. e Confocal images of JC-1 in H1975 cells treated with 4 μM different formulations (dosage based on Fluplatin) for 6 h, and their fluorescence intensity was quantified ( j ), ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. Western blotting analysis of p-elF2α, elF2α, CHOP, and ATF4 in H1975 cells ( f ) and A549 cells ( g ) after treatment with 4 μM of different formulations (dosage based on Fluplatin; n = 3 independent samples). h Western blotting analysis of p-elF2α, elF2α, CHOP, and ATF4 in H1975 cells after treatment with different concentration of FP NPs for 12 h ( n = 3 independent samples). i Confocal images of IF staining against γ-H2AX in H1975 cells treated with 4 μM FP NPs for 6 h, and their fluorescence intensity was quantified ( k ), ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. a Created with BioRender.com. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a file.

Article Snippet: The p53 mutant human NSCLC cell lines NCI-H1975 (American Type Culture Collection (ATCC), #CRL-5908), NCI-H2087 (ATCC, #CRL-5922), NCI-H2342 (ATCC, #CRL-5941), NCI-H1793 (ATCC, #CRL-5896), NCI-H2196 (ATCC, #CRL-5932), NCI-H748 (ATCC, #CRL-5841), NCI-H1770 (ATCC, #CRL-5893), NCI-H1591 (ATCC, #CRL-5878), and NCI-H520 (ATCC, #HTB-182) were purchased from ATCC.

Techniques: Fluorescence, Western Blot, Concentration Assay, Staining

Journal: Nature Communications

Article Title: Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer

doi: 10.1038/s41467-024-47080-3

Figure Lengend Snippet: a Western blotting analysis of p53 in cells after treatment with 4 μM of different formulations (dosage based on Fluplatin) for 12 h. Their grayscale values were quantified ( b – e ) ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). f Western blotting analysis of p53 in cells after treatment with different concentrations of FP NPs for 12 h. Their grayscale values were quantified ( g , h ) ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). i Western blotting analysis of p53 in cells after treatment with 4 μM of FP NPs for 12 h. And their grayscale values were quantified ( j ) ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). k Western blot of cells treated with 4 μM FP NPs or not for 12 h and cultured in medium containing 10 μM MG132 or 10 mM 3-MA. Their grayscale values were quantified ( l ) ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Western blot of H1975 ( m ), H2087 ( n ), H2342 ( o ), and A549 ( p ) cells treated with 2 μM FP NPs or not for 12 h and then treated with 100 μg/mL CHX at the indicated time points. n = 3 biologically independent samples, relative p53/actin ratios are shown. q Western blotting analysis of total ubiquitination (Ub) of immunoprecipitated (IP) p53 in cells after treatment with 2 μM FP NPs with or without MG132 ( n = 3 independent samples). r HMGR inhibition assay under different formulations ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). s The volcano plot shows differential expression of FP NPs treated and untreated H1975 cells. t Schematic summary of the p53-related antitumor mechanism of FP NPs. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a file.

Article Snippet: The p53 mutant human NSCLC cell lines NCI-H1975 (American Type Culture Collection (ATCC), #CRL-5908), NCI-H2087 (ATCC, #CRL-5922), NCI-H2342 (ATCC, #CRL-5941), NCI-H1793 (ATCC, #CRL-5896), NCI-H2196 (ATCC, #CRL-5932), NCI-H748 (ATCC, #CRL-5841), NCI-H1770 (ATCC, #CRL-5893), NCI-H1591 (ATCC, #CRL-5878), and NCI-H520 (ATCC, #HTB-182) were purchased from ATCC.

Techniques: Western Blot, Cell Culture, Ubiquitin Proteomics, Immunoprecipitation, Inhibition, Quantitative Proteomics

Journal: Nature Communications

Article Title: Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer

doi: 10.1038/s41467-024-47080-3

Figure Lengend Snippet: a Ex vivo biodistribution imaging of the main organs in mice bearing H1975 xenografts of the DiR@FP NPs at various time points post injection. Scale bars, 5 mm. b ICP‒MS measurement of Pt accumulation in individual organs at 1, 6, 12, and 24 h after treatment with cisplatin (i) or FP NPs (ii) ( n = 3 mice per group). c ICP‒MS measurement of Pt accumulation in the tumor tissues at 1, 6, 12, and 24 h after treatment with cisplatin or FP NPs ( n = 3 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). d TC levels in tumor tissues and TC, TG, HDL, LDL levels in serum after treatment with each group ( n = 3 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). Confocal images of ATF4 ( e ) and γ-H2AX ( f ) in the tumor tissues of mice after treatment with each group. Scale bars, 100 μm. g Western blotting analysis of p53 R273H in tumor tissues after treatment with each group. Their grayscale values were quantified ( h ) ( n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). i IHC of p53 in the tumor tissues after treatment with each group. Scale bars, 20 μm. j Kaplan-Meier survival curve of mice treated with each group over 70 days ( n = 8 mice per group; Log-rank Mantel–Cox test). k The body weight of mice during treatments with each group over 70 days ( n = 8 mice per group;two-way ANOVA followed by Tukey’s multiple comparisons post test). l IHC of Ki67 in the tumor tissues after treatment with each group. Scale bars, 20 μm. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a file.

Article Snippet: The p53 mutant human NSCLC cell lines NCI-H1975 (American Type Culture Collection (ATCC), #CRL-5908), NCI-H2087 (ATCC, #CRL-5922), NCI-H2342 (ATCC, #CRL-5941), NCI-H1793 (ATCC, #CRL-5896), NCI-H2196 (ATCC, #CRL-5932), NCI-H748 (ATCC, #CRL-5841), NCI-H1770 (ATCC, #CRL-5893), NCI-H1591 (ATCC, #CRL-5878), and NCI-H520 (ATCC, #HTB-182) were purchased from ATCC.

Techniques: Ex Vivo, Imaging, Injection, Western Blot

Journal: Nature Communications

Article Title: Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer

doi: 10.1038/s41467-024-47080-3

Figure Lengend Snippet: a Schematic illustration of the experimental design. b The tumor volumes of mice during treatments with each group ( n = 7 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). c The body weight of mice during treatments with each group ( n = 7 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). d Tumor volume changes for each mouse in each group ( n = 7 mice per group). e In vivo bioluminescence imaging of tumor-bearing mice receiving various treatments after surgery. Three representative mice in each treatment group are shown. Images of day 0 were taken on the day of surgery. f The final anatomical picture and H&E staining of the lungs ( n = 3 independent samples; Scale bars, 5 mm). g IHC of p53, E-cadherin, MMP-2, MMP-9, Vimentin in the primary tumor tissues of cisplatin or FP NPs treatment, and recurrent and metastatic tumor tissues of cisplatin treatment ( n = 3 independent samples). Scale bars, 20 μm. h Recurrence tumor weights of different groups on day 42 after surgery ( n = 7 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). i Kaplan-Meier survival curve of mice treated with each group over 65 days ( n = 7 mice per group; Log-rank Mantel–Cox test). j Final weights of the heart, liver, spleen, lungs, and kidneys ( n = 7 mice per group;two-tailed unpaired t test). k Heatmap of TNF-α, VEGF, IL-10 and IL-6 expression profiles in serum ( n = 3 mice per group). l Indicators of routine blood examination ( n = 3 mice per group; two-tailed unpaired t test) of mice. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a file.

Article Snippet: The p53 mutant human NSCLC cell lines NCI-H1975 (American Type Culture Collection (ATCC), #CRL-5908), NCI-H2087 (ATCC, #CRL-5922), NCI-H2342 (ATCC, #CRL-5941), NCI-H1793 (ATCC, #CRL-5896), NCI-H2196 (ATCC, #CRL-5932), NCI-H748 (ATCC, #CRL-5841), NCI-H1770 (ATCC, #CRL-5893), NCI-H1591 (ATCC, #CRL-5878), and NCI-H520 (ATCC, #HTB-182) were purchased from ATCC.

Techniques: In Vivo, Imaging, Staining, Two Tailed Test, Expressing

Journal: The Journal of investigative dermatology

Article Title: Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

doi: 10.1038/jid.2008.208

Figure Lengend Snippet: Figure 2. Effects of low and high UVB doses on gene expression in melanoma cell lines. Time-course western blot analysis of the effect of low (30 mJ/cm2) and high (70 mJ/cm2) UVB doses on the expression levels of p16, p53, and p21Cip1/Waf1 in Malme-3M (a) and SkMel-28 (b) melanoma cell lines. Cell lysates were collected at different time points (6, 12, 24, and 48 hours) post-UVB irradiation and examined for the expression of p16, p53, and p21. Equal protein loading was confirmed by reprobing the membrane for actin expression. ( þ ): UVB irradiated; (): no UVB irradiation.

Article Snippet: Malme-3M (wild-type p53 and p16) and SkMel-28 (mutant p53 and wild-type p16) human melanoma cell lines were purchased from ATCC (Manassas, Virginia) and cultured in RPMI 1640 media containing 10% (v/v) fetal bovine serum.

Techniques: Gene Expression, Western Blot, Expressing, Irradiation, Membrane

Journal: The Journal of investigative dermatology

Article Title: Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

doi: 10.1038/jid.2008.208

Figure Lengend Snippet: Figure 3. Reestablishment of p16 pathway in HaCaT cells and its effect on cell-cycle and apoptosis mechanisms, in response to both low and high doses of UVB irradiation. (a) Time-course western blot analysis of the effect of low (30 mJ/cm2) and high (70 mJ/cm2) UVB doses on the expression levels of p53 and its downstream target gene associated with cell cycle (p21) as well as apoptosis (Bax and Bcl-2) in HaCaT cells 6, 12, 24, and 48 hours post-UVB; (b) effects of low (30 mJ/cm2) and high (70 mJ/cm2) UVB doses on the expression levels of p53 and p21 following transient transfection (Tx) of p16 to HaCaT cells. Equal protein loading was confirmed by reprobing the membrane for actin expression. ( þ ): UVB irradiated; (): not irradiated; ( þ C): lysates collected from high-dose UVB-irradiated SkMel-28 cells at 24 hours are used as a positive control for p16 expression. (C): No UVB irradiation. Equal protein loading was confirmed by reprobing the membrane for b-tubulin expression.

Article Snippet: Malme-3M (wild-type p53 and p16) and SkMel-28 (mutant p53 and wild-type p16) human melanoma cell lines were purchased from ATCC (Manassas, Virginia) and cultured in RPMI 1640 media containing 10% (v/v) fetal bovine serum.

Techniques: Irradiation, Western Blot, Expressing, Transfection, Membrane, Positive Control

Journal: The Journal of investigative dermatology

Article Title: Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

doi: 10.1038/jid.2008.208

Figure Lengend Snippet: Figure 4. Effect of reinstatement of p16 pathway on cell cycle and apoptosis in UVB-irradiated HaCaT cells. HaCaT cells were irradiated with low (a) and high (b) UVB doses and analyzed by flow cytometry at different time points post-UVB irradiation. On the other hand, HaCaT cells were transiently transfected (TX) with p16 cDNA, irradiated with low (c) and high (d) UVB doses, and analyzed by flow cytometry at different time points (6, 12, 24, 48, and 72 hours) post-UVB irradiation. C (no UV): cells were not irradiated; TX (no UV): cells were transfected with p16 cDNA but did not receive UVB irradiation.

Article Snippet: Malme-3M (wild-type p53 and p16) and SkMel-28 (mutant p53 and wild-type p16) human melanoma cell lines were purchased from ATCC (Manassas, Virginia) and cultured in RPMI 1640 media containing 10% (v/v) fetal bovine serum.

Techniques: Irradiation, Flow Cytometry, Transfection

Journal: The Journal of investigative dermatology

Article Title: Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

doi: 10.1038/jid.2008.208

Figure Lengend Snippet: Figure 5. Effects of low (30 mJ/cm2) and high (70 mJ/cm2) UVB doses on p16 and p53 expression levels in human skin organ cultures. Time-course western blot analysis of p53 and p16 expression in UVB-irradiated human skin organ cultures 6, 12, 24, and 48 hours post-UVB treatment. Equal protein loading was confirmed by reprobing the membrane for b-tubulin expression.

Article Snippet: Malme-3M (wild-type p53 and p16) and SkMel-28 (mutant p53 and wild-type p16) human melanoma cell lines were purchased from ATCC (Manassas, Virginia) and cultured in RPMI 1640 media containing 10% (v/v) fetal bovine serum.

Techniques: Expressing, Western Blot, Irradiation, Membrane

Journal: The Journal of investigative dermatology

Article Title: Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

doi: 10.1038/jid.2008.208

Figure Lengend Snippet: Figure 6. Effects of high (70 mJ/cm2) UVB dose on apoptosis and expression of both p53 and p16 proteins in normal human skin organ cultures 24 and 48 hours post-UVB treatment. (a and b) Hematoxylin and eosin staining of human skin organ cultures 24 hours (a) and 48 hours (b) after UVB irradiation with high dose showing sunburn cells (arrows). (c, d) Time-course analysis of p53 (c, d) by immunohistochemistry showing nuclear staining of p53 protein (arrows) at 24 hours (c) and 48 hours (d) post-UVB irradiation. (e, f) Time- course analysis p16 (e, f) expression by immunohistochemistry showing p16 nuclear and cytoplasmic immunostaining (arrows) at 24 hours (e) and 48 hours (f) post-UVB irradiation. (g, h) p53-immunohistochemistry on adjacent sections of normal skin not exposed to UVB treatment.

Article Snippet: Malme-3M (wild-type p53 and p16) and SkMel-28 (mutant p53 and wild-type p16) human melanoma cell lines were purchased from ATCC (Manassas, Virginia) and cultured in RPMI 1640 media containing 10% (v/v) fetal bovine serum.

Techniques: Expressing, Staining, Irradiation, Immunohistochemistry, Immunostaining