p53r273h expression vectors  (Addgene inc)

Journal: bioRxiv

Article Title: Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis

doi: 10.1101/2024.02.22.581532

Figure Lengend Snippet: A. 20S proteasome activity was determined in the indicated cell lines using a fluorometric proteosome activity assay. All pairwise comparisons were made using student’s t-test. B. (Left) Basal levels of 20S proteasome activity in lysates of H1975 Control and p53 KO cells. ( Right ) Cell lysates of H1975-Control and H1975-p53KO (p53KO) cell lines generated using CRISPR/Cas9 were immunoblotted with p53 and GAPDH antibodies. C. The indicated cells were treated with vehicle or increasing concentrations of bortezomib (BTZ; left) or carfilzomib (CFZ; right) for 72 h, and cell viability was determined by the MTT assay. D . H1975-Control and H1975-p53KO cells were treated with vehicle (-) or BTZ (5 nM) for 48 h and cell viability was determined by Trypan-blue exclusion assay. E. ( Left ) H460 and H460-p53KO cells were treated with vehicle (-) or BTZ (5 nM) for 72 h and cell viability was determined by WST-1 assay. ( Right ) Cell lysates of H460 and H460-p53KO cells were immunoblotted with p53 and GAPDH antibodies. F. ( Left ) H460-p53KO cells stably expressing GFP or p53R273H cDNA were treated with increasing concentrations of BTZ for 72 h and cell viability was determined by WST-1 assay. ( Right ) Cell lysates of H460-p53KO cells stably expressing GFP or p53R273H cDNA were immunoblotted with p53 and GAPDH antibodies. * p <0.05, ** p <0.01, *** p <0.005, ns indicates p >0.05. Error bars indicate +/- 1.0 S.D.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) vectors, shNOXA (TCRN0000338867), shNOXA#5 TCRN0000338864), shNRF2 (TCRN0000273494), shATF3 (TCRN0000329690), and shATF3#3 (TCRN000013568) were purchased from Sigma Aldrich (St. Louis, MO, USA). shControl (1864), GFP (35637) and p53R273H expression vectors (22934) were purchased from Addgene (Watertown, MA, USA).

Techniques: Activity Assay, Control, Generated, CRISPR, MTT Assay, Trypan Blue Exclusion Assay, WST-1 Assay, Stable Transfection, Expressing

Journal: Cellular and molecular life sciences : CMLS

Article Title: Loss of fragile WWOX gene leads to senescence escape and genome instability.

doi: 10.1007/s00018-023-04950-1

Figure Lengend Snippet: Fig. 6 ROS inhibition results in senescence induction in late- passage Wwox−/− MEFs. a ROS levels in Wwox+/+, Wwox+/− and Wwox−/− MEFs at early- and late-passages were determined by DHE staining and flow cyto- metric analysis. b PCR amplifi- cation of mononucleotide repeat markers Bat26, Bat30, Bat37, Bat64 and Bat67 was performed using the genomic DNA sam- ples isolated from NAC-treated late-passage Wwox+/+ and Wwox−/− MEFs, and the length of PCR products was analyzed by capillary electrophoresis for detecting microsatellite insta- bility. c, d SA-β-gal staining was performed in late-passage Wwox+/+, Wwox+/−, Wwox−/−, and 1 mM NAC-treated Wwox−/− MEFs. The representa- tive images obtained from at least three independent experi- ments are shown in d. Scale bars = 100 µm. The percentages of SA-β-gal-positive senescent cells are shown in c. *P ≤ 0.05; ***P ≤ 0.005; Two-tailed t test. e Cellular morphology of Wwox+/+, Wwox+/−, Wwox−/−, and 1 mM NAC-treated Wwox−/− MEFs at early- and late-passages was examined. Scale bars = 100 µm. f DNA methylation of p16Ink4a pro- moter at specific CpG islands was analyzed. N.S. not sig- nificant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005; Two-tailed t test. g p16Ink4a, p21Cip1/Waf1, p53 and WWOX protein expression was examined in late-passage Wwox+/−, Wwox−/−, and 1 mM NAC-treated Wwox−/− MEFs. β-actin was used as an internal control in western blotting

Article Snippet: Human p21Cip1/Waf1 and p53 expression vectors were obtained from Addgene (#20814 and #11770, respectively; Watertown, MA, USA).

Techniques: Inhibition, Staining, Isolation, Electrophoresis, Two Tailed Test, DNA Methylation Assay, Expressing, Control, Western Blot

Journal: PLoS ONE

Article Title: LOC401317, a p53-Regulated Long Non-Coding RNA, Inhibits Cell Proliferation and Induces Apoptosis in the Nasopharyngeal Carcinoma Cell Line HNE2

doi: 10.1371/journal.pone.0110674

Figure Lengend Snippet: HNE2 cells were transfected with a TP53 expression vector (the pCMV-p53 plasmid). Levels of ( A ) TP53 mRNA transcripts and ( B ) p53 protein expression were determined at 0–48 h post-transfection by qRT-PCR and western blotting, respectively. ( C ) To measure p53 transcriptional activity, the pCMV-p53 and the pp53-TA-luc plasmids were cotransfected into HNE2 cells, and transcriptional activity of p53 from 0–48 h post-transfection was determined by luciferase assays. Similarly, induction of ( D ) MDM2 mRNA transcripts and ( E ) MDM2 protein expression in HNE2 were also determined in HNE2 pCMV-p53 transfectants by qRT-PCR and western blotting. GAPDH protein expression was detected as a loading control for p53 and MDM2 western blots. Data shown are representative of 3 independent experiments. Bar graphs show mean ± S.D. ** P <0.01, *** P <0.001.

Article Snippet: The p53 expression vector, pCMV-p53, was purchased from Clontech Laboratories Inc. (Mountain view, CA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase

Journal: PLoS ONE

Article Title: LOC401317, a p53-Regulated Long Non-Coding RNA, Inhibits Cell Proliferation and Induces Apoptosis in the Nasopharyngeal Carcinoma Cell Line HNE2

doi: 10.1371/journal.pone.0110674

Figure Lengend Snippet: ( A ) A total of 133 lncRNAs were upregulated in HNE2 cells by more than 2.0-fold in at least one time point (12, 24, or 48 h post-pCMV-p53 transfection), while ( B ) 1057 lncRNAs were downregulated by more than 2.0-fold in at least 1 time point in HNE2 cells. ( C ) Detailed expression profiles of the top 30 lncRNAs that were the most significantly upregulated by TP53 transgene expression. ( D ) Validation of 5 of the top 30 most-upregulated lncRNAs in HNE2 by qRT-PCR. Data shown reflect the means of 3 independent experiments ± S.D. * P <0.05, ** P <0.01, *** P <0.001. Expression data are exhibited as normalized log ratios to control time points (0 h) for each lncRNA. ( E ) Validation of LOC401317 expression in HNE1 and CNE2 cells following TP53 transfection by qRT-PCR. Data shown reflect the mean of 3 independent experiments ± S.D. ** P <0.01, *** P <0.001. Expression data were normalized to control time points (0 h).

Article Snippet: The p53 expression vector, pCMV-p53, was purchased from Clontech Laboratories Inc. (Mountain view, CA, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: LOC401317, a p53-Regulated Long Non-Coding RNA, Inhibits Cell Proliferation and Induces Apoptosis in the Nasopharyngeal Carcinoma Cell Line HNE2

doi: 10.1371/journal.pone.0110674

Figure Lengend Snippet: ( A ) Schematic representation of the potential promoter and p53-binding site upstream of LOC401317 transcription start site. Wild-type and mutated versions of the putative p53-binding site are indicated. ( B ) Transactivation of the putative LOC401317 promoter by p53. An 820-bp sequence, −570 bp to +249 bp to the transcript start site of LOC401317, containing the potential promoter and p53-binding site was inserted into the pGL3 luciferase reporter vector (LOC-promoter). The empty pGL3 vector served as a negative control (pGL3-EV). Luciferase vectors were cotransfected with pCMV-p53 or the parental vector (Vector) into HNE2 cells, as indicated. Cells were then harvested for luciferase assays 24 h after transfection. ( C ) Activation of the potential p53-binding site adjacent to the LOC401317 promoter by p53. Synthetic oligonucleotides containing 3 tandem wild-type p53-binding sites (LOC-WT) or mutant p53-binding sites (LOC-MT, negative control) were inserted into the luciferase reporter vector. The pp53-TA-luc vector, which contains conserved p53-binding sites, was used as a positive control. Luciferase vectors were cotransfected with pCMV-p53 or the parental vector into HNE2 cells, as shown. Cells were harvested for luciferase assays 24 h after transfection. The results showed that p53 significantly induces luciferase activity (>2.76-fold) via the wild-type p53-binding site. Data shown are the means of 3 independent experiments ± S.D. ** P <0.01.

Article Snippet: The p53 expression vector, pCMV-p53, was purchased from Clontech Laboratories Inc. (Mountain view, CA, USA).

Techniques: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Negative Control, Transfection, Activation Assay, Mutagenesis, Positive Control, Activity Assay