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p53r273h expression vectors  (Addgene inc)


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    Addgene inc p53r273h expression vectors
    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 <t>p53R273H</t> 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.
    P53r273h Expression Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/p53+expression+vector/pLenti6%2FV5-p53_R273H+(Plasmid+%2322934)/bio_rxiv__2024__02__22__581532-154-30-37
    Average 93 stars, based on 22 article reviews
    p53r273h expression vectors - by Bioz Stars, 2026-10
    93/100 stars

    Images

    1) Product Images from "Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis"

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

    Journal: bioRxiv

    doi: 10.1101/2024.02.22.581532

    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.
    Figure Legend 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.

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

    Related Articles

    Expressing:

    Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis
    Article Snippet: .. The p53 expression vector (pHMT.wt p53) was a gift from James Sherley (catalog# 12139, Addgene plasmid, Boston, MA, USA). ..

    Article Title: MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway
    Article Snippet: Simultaneously, HepG 2 cells cultured with the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO (40 nM) or miR-21-MMASO (control) (Both Exiqon). .. Lipid accumulation was determined by Nile-Red Staining followed by microfluorimeter detection or imaging. p53 expression vector (Plasmid #: 12091) and p53 and Hbp1 siRNA expression vectors were purchased from Addgene and Origene, respectively; and HBP1 expression vector (pPM-hHBP1His vector) was purchased from Applied Biological Materials (ABM) Inc. To determine whether the inhibitory effect of miR-21-ASO on lipogenesis is mediated by HBP1, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO ( 40 nM in 4-well chamber slides), or miR-21-ASO combined with HBP1 siRNA expression vector (200 ng/well in 4-well chamber slides). ..

    Article Title: p53 regulates the expression of human angiotensin II AT 2 receptor interacting protein (ATIP1) gene
    Article Snippet: .. To test the effect of p53 on ATIP1 expression, a p53 expression vector (Addgene plasmid 10838, Addgene Inc., Cambridge, MA, USA), empty pcDNA3.0 vector (Invitrogen), siRNA specific to p53 (Cell Signaling, MA, USA) or control siRNA were transfected as previously described ( 11 ). .. To test the effect of DNA damage on ATIP1 expression, 50 nm of cisplatin (cis‐diamminedichloroplatinum II; Sigma‐Aldrich, St. Louis, MO, USA) or vehicle alone was incubated with the WI38 cells for 4 h. Total RNA from the cells was isolated using an RNeasy mini kit (Qiagen, Germany).

    Article Title: Elevated PRC1 in gastric carcinoma exerts oncogenic function and is targeted by piperlongumine in a p53‐dependent manner
    Article Snippet: .. The resultant promoter reporter plasmids were generated by inserting the serial deleted fragments of the 5‐flanking region of PRC1 promoter upstream of the initiating ATG into pGL3‐basic vector digested with KpnI and XhoI in the sense orientation. p53 expression vector (pcDNA3.1‐p53) was constructed by Dr. Thomas Roberts , and empty vector (pcDNA3.1) was purchased from Addgene. siRNAs against p53 (ONTARGETplus SMARTpool Tp53 siRNA) and the non‐targeting siRNAs (Control‐siRNA) were purchased from GE Healthcare Dharmacon Inc.(Lafayette, CO, USA). .. PRC1 (sc‐8356), PARP (sc‐7150), GADD45α (sc‐797) and p53 (sc‐6243) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Article Title: p53 regulates the expression of human angiotensin II AT 2 receptor interacting protein (ATIP1) gene
    Article Snippet: .. We thank Dr Thomas Roberts from Dana‐Farber Cancer Institute for providing us with the p53 expression vector through Addgene Inc. We thank Ms. Katherine Long for her editorial assistance. ..

    Plasmid Preparation:

    Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis
    Article Snippet: .. The p53 expression vector (pHMT.wt p53) was a gift from James Sherley (catalog# 12139, Addgene plasmid, Boston, MA, USA). ..

    Article Title: MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway
    Article Snippet: Simultaneously, HepG 2 cells cultured with the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO (40 nM) or miR-21-MMASO (control) (Both Exiqon). .. Lipid accumulation was determined by Nile-Red Staining followed by microfluorimeter detection or imaging. p53 expression vector (Plasmid #: 12091) and p53 and Hbp1 siRNA expression vectors were purchased from Addgene and Origene, respectively; and HBP1 expression vector (pPM-hHBP1His vector) was purchased from Applied Biological Materials (ABM) Inc. To determine whether the inhibitory effect of miR-21-ASO on lipogenesis is mediated by HBP1, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO ( 40 nM in 4-well chamber slides), or miR-21-ASO combined with HBP1 siRNA expression vector (200 ng/well in 4-well chamber slides). ..

    Article Title: p53 regulates the expression of human angiotensin II AT 2 receptor interacting protein (ATIP1) gene
    Article Snippet: .. To test the effect of p53 on ATIP1 expression, a p53 expression vector (Addgene plasmid 10838, Addgene Inc., Cambridge, MA, USA), empty pcDNA3.0 vector (Invitrogen), siRNA specific to p53 (Cell Signaling, MA, USA) or control siRNA were transfected as previously described ( 11 ). .. To test the effect of DNA damage on ATIP1 expression, 50 nm of cisplatin (cis‐diamminedichloroplatinum II; Sigma‐Aldrich, St. Louis, MO, USA) or vehicle alone was incubated with the WI38 cells for 4 h. Total RNA from the cells was isolated using an RNeasy mini kit (Qiagen, Germany).

    Article Title: p53 regulates the expression of human angiotensin II AT 2 receptor interacting protein (ATIP1) gene
    Article Snippet: .. We thank Dr Thomas Roberts from Dana‐Farber Cancer Institute for providing us with the p53 expression vector through Addgene Inc. We thank Ms. Katherine Long for her editorial assistance. ..

    Staining:

    Article Title: MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway
    Article Snippet: Simultaneously, HepG 2 cells cultured with the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO (40 nM) or miR-21-MMASO (control) (Both Exiqon). .. Lipid accumulation was determined by Nile-Red Staining followed by microfluorimeter detection or imaging. p53 expression vector (Plasmid #: 12091) and p53 and Hbp1 siRNA expression vectors were purchased from Addgene and Origene, respectively; and HBP1 expression vector (pPM-hHBP1His vector) was purchased from Applied Biological Materials (ABM) Inc. To determine whether the inhibitory effect of miR-21-ASO on lipogenesis is mediated by HBP1, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO ( 40 nM in 4-well chamber slides), or miR-21-ASO combined with HBP1 siRNA expression vector (200 ng/well in 4-well chamber slides). ..

    Imaging:

    Article Title: MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway
    Article Snippet: Simultaneously, HepG 2 cells cultured with the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO (40 nM) or miR-21-MMASO (control) (Both Exiqon). .. Lipid accumulation was determined by Nile-Red Staining followed by microfluorimeter detection or imaging. p53 expression vector (Plasmid #: 12091) and p53 and Hbp1 siRNA expression vectors were purchased from Addgene and Origene, respectively; and HBP1 expression vector (pPM-hHBP1His vector) was purchased from Applied Biological Materials (ABM) Inc. To determine whether the inhibitory effect of miR-21-ASO on lipogenesis is mediated by HBP1, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO ( 40 nM in 4-well chamber slides), or miR-21-ASO combined with HBP1 siRNA expression vector (200 ng/well in 4-well chamber slides). ..

    Cell Culture:

    Article Title: MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway
    Article Snippet: Simultaneously, HepG 2 cells cultured with the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO (40 nM) or miR-21-MMASO (control) (Both Exiqon). .. Lipid accumulation was determined by Nile-Red Staining followed by microfluorimeter detection or imaging. p53 expression vector (Plasmid #: 12091) and p53 and Hbp1 siRNA expression vectors were purchased from Addgene and Origene, respectively; and HBP1 expression vector (pPM-hHBP1His vector) was purchased from Applied Biological Materials (ABM) Inc. To determine whether the inhibitory effect of miR-21-ASO on lipogenesis is mediated by HBP1, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO ( 40 nM in 4-well chamber slides), or miR-21-ASO combined with HBP1 siRNA expression vector (200 ng/well in 4-well chamber slides). ..

    Transfection:

    Article Title: MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway
    Article Snippet: Simultaneously, HepG 2 cells cultured with the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO (40 nM) or miR-21-MMASO (control) (Both Exiqon). .. Lipid accumulation was determined by Nile-Red Staining followed by microfluorimeter detection or imaging. p53 expression vector (Plasmid #: 12091) and p53 and Hbp1 siRNA expression vectors were purchased from Addgene and Origene, respectively; and HBP1 expression vector (pPM-hHBP1His vector) was purchased from Applied Biological Materials (ABM) Inc. To determine whether the inhibitory effect of miR-21-ASO on lipogenesis is mediated by HBP1, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with miR-21-ASO ( 40 nM in 4-well chamber slides), or miR-21-ASO combined with HBP1 siRNA expression vector (200 ng/well in 4-well chamber slides). ..

    Article Title: p53 regulates the expression of human angiotensin II AT 2 receptor interacting protein (ATIP1) gene
    Article Snippet: .. To test the effect of p53 on ATIP1 expression, a p53 expression vector (Addgene plasmid 10838, Addgene Inc., Cambridge, MA, USA), empty pcDNA3.0 vector (Invitrogen), siRNA specific to p53 (Cell Signaling, MA, USA) or control siRNA were transfected as previously described ( 11 ). .. To test the effect of DNA damage on ATIP1 expression, 50 nm of cisplatin (cis‐diamminedichloroplatinum II; Sigma‐Aldrich, St. Louis, MO, USA) or vehicle alone was incubated with the WI38 cells for 4 h. Total RNA from the cells was isolated using an RNeasy mini kit (Qiagen, Germany).

    Control:

    Article Title: p53 regulates the expression of human angiotensin II AT 2 receptor interacting protein (ATIP1) gene
    Article Snippet: .. To test the effect of p53 on ATIP1 expression, a p53 expression vector (Addgene plasmid 10838, Addgene Inc., Cambridge, MA, USA), empty pcDNA3.0 vector (Invitrogen), siRNA specific to p53 (Cell Signaling, MA, USA) or control siRNA were transfected as previously described ( 11 ). .. To test the effect of DNA damage on ATIP1 expression, 50 nm of cisplatin (cis‐diamminedichloroplatinum II; Sigma‐Aldrich, St. Louis, MO, USA) or vehicle alone was incubated with the WI38 cells for 4 h. Total RNA from the cells was isolated using an RNeasy mini kit (Qiagen, Germany).

    Article Title: Elevated PRC1 in gastric carcinoma exerts oncogenic function and is targeted by piperlongumine in a p53‐dependent manner
    Article Snippet: .. The resultant promoter reporter plasmids were generated by inserting the serial deleted fragments of the 5‐flanking region of PRC1 promoter upstream of the initiating ATG into pGL3‐basic vector digested with KpnI and XhoI in the sense orientation. p53 expression vector (pcDNA3.1‐p53) was constructed by Dr. Thomas Roberts , and empty vector (pcDNA3.1) was purchased from Addgene. siRNAs against p53 (ONTARGETplus SMARTpool Tp53 siRNA) and the non‐targeting siRNAs (Control‐siRNA) were purchased from GE Healthcare Dharmacon Inc.(Lafayette, CO, USA). .. PRC1 (sc‐8356), PARP (sc‐7150), GADD45α (sc‐797) and p53 (sc‐6243) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Generated:

    Article Title: Elevated PRC1 in gastric carcinoma exerts oncogenic function and is targeted by piperlongumine in a p53‐dependent manner
    Article Snippet: .. The resultant promoter reporter plasmids were generated by inserting the serial deleted fragments of the 5‐flanking region of PRC1 promoter upstream of the initiating ATG into pGL3‐basic vector digested with KpnI and XhoI in the sense orientation. p53 expression vector (pcDNA3.1‐p53) was constructed by Dr. Thomas Roberts , and empty vector (pcDNA3.1) was purchased from Addgene. siRNAs against p53 (ONTARGETplus SMARTpool Tp53 siRNA) and the non‐targeting siRNAs (Control‐siRNA) were purchased from GE Healthcare Dharmacon Inc.(Lafayette, CO, USA). .. PRC1 (sc‐8356), PARP (sc‐7150), GADD45α (sc‐797) and p53 (sc‐6243) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Construct:

    Article Title: Elevated PRC1 in gastric carcinoma exerts oncogenic function and is targeted by piperlongumine in a p53‐dependent manner
    Article Snippet: .. The resultant promoter reporter plasmids were generated by inserting the serial deleted fragments of the 5‐flanking region of PRC1 promoter upstream of the initiating ATG into pGL3‐basic vector digested with KpnI and XhoI in the sense orientation. p53 expression vector (pcDNA3.1‐p53) was constructed by Dr. Thomas Roberts , and empty vector (pcDNA3.1) was purchased from Addgene. siRNAs against p53 (ONTARGETplus SMARTpool Tp53 siRNA) and the non‐targeting siRNAs (Control‐siRNA) were purchased from GE Healthcare Dharmacon Inc.(Lafayette, CO, USA). .. PRC1 (sc‐8356), PARP (sc‐7150), GADD45α (sc‐797) and p53 (sc‐6243) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).



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    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 <t>p53R273H</t> 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.
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    Millipore construct for lentiviral vector (plko.1) expressing control shrna (shp002) and a shrna against mouse p53 gene (trp53) (cat. trcn0000173949)
    HNE2 cells were transfected with a TP53 expression vector (the <t>pCMV-p53</t> plasmid). Levels of ( A ) TP53 mRNA transcripts and ( B ) <t>p53</t> <t>protein</t> 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.
    Construct For Lentiviral Vector (Plko.1) Expressing Control Shrna (Shp002) And A Shrna Against Mouse P53 Gene (Trp53) (Cat. Trcn0000173949), supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    construct for lentiviral vector (plko.1) expressing control shrna (shp002) and a shrna against mouse p53 gene (trp53) (cat. trcn0000173949) - by Bioz Stars, 2026-10
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    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.

    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

    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

    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

    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.

    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

    ( 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).

    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

    ( 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.

    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