pp53-ta-luc reporter plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: DNA repair and anti-cancer mechanisms in the longest-living mammal: the bowhead whale

doi: 10.1101/2023.05.07.539748

Figure Lengend Snippet: a , Images of representative fibroblast colonies for tested cell lines after 4 weeks of growth in soft agar. The top panel indicates whether the cell lines in the column below have the indicated protein overexpressed (+), inactivated (-), or expressed in the active endogenous form (WT). Text above individual images indicate for that cell line whether tumor suppressors are inactivated through genetic knockout or SV40 Large T (or LT mutants) or Small T (ST) antigen. Icons in corners of images indicate species. Scale bar is 250 µm. b , Volumetric growth curves for the indicated bowhead whale fibroblast cell lines in mouse xenograft assays. All cell lines shown stably express H-Ras G12V and hTERT in addition to the genotype indicated in the figure legend. Data points represent averages from 3 immunodeficient nude mice injected bilaterally (6 injections) for each cell line, except for TP53 -/- RB1 -/- double knockouts, for which 2 independent cell lines were tested, for a total of 6 mice/12 injections. Experiments were terminated based on endpoints for maximum tumor size or duration of experiment as described in Methods. Images in the legend show a representative mouse for the indicated cell line at the final measured time point. Error bars show SEM. c , Western blot for p53 protein in clonally isolated fibroblast colonies following CRISPR targeting of TP53 . Underlined lanes indicate colonies selected for further validation and experiments. d , Western blot for Rb protein in fibroblast clones following CRISPR targeting of RB1 on an p53 knockout background.

Article Snippet: For p53 activity measurement, 1 x 10 cells of control (WT) and clonally isolated p53 KO cell lines were electroporated with 3 µg p53 firefly luciferase reporter plasmid pp53-TA-Luc (Clontech/Takara) and 0.3 ug renilla luciferase control plasmid pRL-CMV (Promega) on an Amaxa Nucleofector 2b (Lonza).

Techniques: Knock-Out, Stable Transfection, Injection, Western Blot, Isolation, CRISPR, Clone Assay

Journal: bioRxiv

Article Title: DNA repair and anti-cancer mechanisms in the longest-living mammal: the bowhead whale

doi: 10.1101/2023.05.07.539748

Figure Lengend Snippet: a , Western blot for p53 protein in fibroblasts clones following CRISPR targeting of TP53 . Labeled clones were for further validation and experiments. b , Western blot for Rb protein in fibroblast clones following CRISPR targeting of RB1 on p53 knockout background. c , Ratio of firefly:renilla luciferase luminescence in fibroblasts transfected with firefly luciferase reporter of p53 transcriptional activity and renilla luciferase control. Cells were treated with etoposide to induce p53 activity. d , Ratio of firefly:renilla luciferase luminescence in fibroblasts transfected with firefly luciferase reporter of E2F transcriptional activity and renilla luciferase control. Transfected cells were serum starved for 24h and returned to complete medium for 24h before luminescence measurement. Higher E2F activity results from reduced Rb activity. Error bars represent SD. ****p<0.001 (two-tailed t test), n=3.

Article Snippet: For p53 activity measurement, 1 x 10 cells of control (WT) and clonally isolated p53 KO cell lines were electroporated with 3 µg p53 firefly luciferase reporter plasmid pp53-TA-Luc (Clontech/Takara) and 0.3 ug renilla luciferase control plasmid pRL-CMV (Promega) on an Amaxa Nucleofector 2b (Lonza).

Techniques: Western Blot, Clone Assay, CRISPR, Labeling, Knock-Out, Luciferase, Transfection, Activity Assay, Two Tailed Test

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: MAGEA3 interacts with KAP1 and inhibits p53 transcriptional activity. A. Co-IP assays were performed in HeLa cells and SiHa cells cotransfected with Lv-MAGEA3 and pcDNA3.1-KAP1. Cell lysates were immunoprecipitated (IP) with control IgG or the indicated antibody, and the precipitated protein was detected by immunoblotting (IB) analysis with the indicated antibody. Cell extracts were used as a positive control (Input). B. Effects of MAGEA3 or KAP1 on p53 activity, as indicated by the reporter plasmid pp53-TA-luc. HeLa cells (with endogenous wild-type p53) grown on a 12-well plate were cotransfected with the pp53-TA-luc and the indicated plasmids. Luciferase was measured at 48 h posttransfection and normalized according to Renilla luciferase activities. Data (means ± SD) are represented as fold differences relative to that observed in cells without transfecting the indicated plasmids. C, D. Western blotting analysis of the p53 and KAP1 proteins following overexpression or knockdown of MAGEA3 in SiHa (Ca, Cb) and HeLa (Da, Db) cells. *P<0.05, **P<0.01 vs. control groups. E. Immunohistochemical analysis of p53 expression in the Lv-MAGEA3 group and Lv-NC group of xenograft tumors. Bar length: 100 µm.

Article Snippet: Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Positive Control, Plasmid Preparation, Luciferase, Over Expression, Knockdown, Immunohistochemical staining, Expressing

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: Effects of MAGEA3 expression on the protein levels of p53 downstream targets. A, B. Western blotting analysis of p53, p21, Bax, Bcl2, PUMA, cyclin D1, and cleaved caspase-3 proteins following overexpression or knockdown of MAGEA3 in SiHa (Aa, Ab) and HeLa (Ba, Bb) cells. *P<0.05, **P<0.01 vs. control groups. C. Immunohistochemical analysis of p21 and Bax expression in the Lv-MAGEA3 group and Lv-NC group of xenograft tumors. Bar length: 100 µm.

Article Snippet: Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Control, Immunohistochemical staining

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: Nutlin3 reverses the effects of MAGEA3 overexpression on the p53 signaling pathway, cell cycle, and apoptosis. A. Flow cytometric analysis of the cell cycle and apoptosis after treatment with Nutlin3 (10 μM) for 48 h. B. Western blotting analysis of the indicated proteins after treatment with Nutli3 (10 μM) for 48 h. *P<0.05, **P<0.01 vs. Lv-NC; ##P<0.01 vs. Lv-MAGEA3.

Article Snippet: Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Over Expression, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Epstein–Barr virus miR-BART3-3p promotes tumorigenesis by regulating the senescence pathway in gastric cancer

doi: 10.1074/jbc.RA118.006853

Figure Lengend Snippet: EBV BART3-3p inhibits p53 expression by targeting its CDS region. A, Western blot analysis of p53 and p21 protein expression in SGC7901 and AGS cells transfected with nc mimics or EBV BART3-3p mimics (b3-3p) for 48 h. B, SGC7901 and AGS cells were cotransfected with GFP-p53 and mimics (b3-3p or nc) for 48 h, and exogenous and endogenous p53 was analyzed by Western blotting. C, KATOIII cells were cotransfected with GFP-p53 and mimics (b3-3p or nc) for 48 h. The Western blot shows p53. D, gene expression levels of TP53, BAX, and CDKN1A in SGC7901 cells transfected with mimics (b3-3p or nc) for 48 h were examined by RT-qPCR (n = 3). Expression levels were normalized to nc. E, BART3-3p regulates p53 transcriptional activity. SGC7901 cells were cotransfected with 200 ng of p53-responsive reporter pp53-TA-Luc plasmid, 50 ng of Renilla plasmid, and 20 pmol of mimics (b3-3p or nc) (n = 3). Luciferase activity was measured 48 h later, and the data are shown as the relative firefly luciferase activity normalized to the value of Renilla luciferase. F, HEK293 cells were cotransfected with luciferase reporters carrying TP53 3′-UTR (or CDS) and mimics (b3-3p or nc) (n = 4). 48 h later, luciferase activity was measured. The data are shown as the relative firefly luciferase activity normalized to the value of Renilla luciferase. G, BART3-3p and its putative binding sequences in the CDS region of TP53. Mutants of the CDS of TP53 gene were generated in the complementary sites that bind to the seed regions of BART3-3p. H, HEK293 cells were cotransfected with luciferase reporters carrying either the predicted miRNA target site in TP53 CDS (WT) or its corresponding mutants (Mut1 and Mut2) and mimics (b3-3p or nc). Luciferase activity was measured 48 h later. The data are shown as the relative firefly luciferase activity normalized to the value of Renilla luciferase. I, SGC7901 cells were transfected with mimics (b3-3p or nc) for 48 h, and cell lysates were immunoprecipitated (IP) using either anti-Ago2 antibody or a negative control IgG. The mRNA levels of TP53 binding to Ago2 were detected by RT-qPCR (n = 4). n represents biologically independent samples, and data are shown as the mean ± 95% CI limit. Statistical significance relative to control was assessed by unpaired two-tailed Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the control group (nc). n.s., not significant. Error bars represent 95% CI. GAPDH was used for protein loading control. Actin was used for normalizing the expression of mRNAs.

Article Snippet: The pp53-TA-luc luciferase reporter plasmid was purchased from Beyotime (Jiangsu, China) to monitor the transcriptional activity of p53.

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Activity Assay, Plasmid Preparation, Luciferase, Binding Assay, Generated, Immunoprecipitation, Negative Control, Two Tailed Test