Journal: OncoTargets and therapy

Article Title: Butein activates p53 in hepatocellular carcinoma cells via blocking MDM2-mediated ubiquitination

doi: 10.2147/OTT.S160119

Figure Lengend Snippet: Butein promoted p53 transcriptional activity. Notes: ( A ) HepG2 cells were transfected with p53 reporter gene and treated with butein for 24 h, and the luciferase activities were examined. * p <0.05, versus the control. ( B ) HepG2 cells were treated with butein for 24 h and the expressions of p53 and Bax were examined.

Article Snippet: The p53 firefly luciferase reporter plasmid (pp53-TA-luc; Beyotime Biotechnology) and the internal control Renilla luciferase reporter plasmid (Promega) were cotransfected into HepG2 using Lipofectamine 2000 according to supplier’s directions.

Techniques: Activity Assay, Transfection, Luciferase, Control

Journal: OncoTargets and therapy

Article Title: Butein activates p53 in hepatocellular carcinoma cells via blocking MDM2-mediated ubiquitination

doi: 10.2147/OTT.S160119

Figure Lengend Snippet: Butein inhibited MDM2-mediated p53 ubiquitination. Notes: ( A ) Butein suppressed the binding of p53 to MDM2. After treatment with 30 μM butein for 24 h, WCE was prepared using the NP40 CO-IP buffer. The cell lysate was incubated with anti-p53 antibody and agarose A/G beads (50% slurry) overnight at 4°C. The supernatant was discarded and the beads were washed using wash buffer 3 times. After boiling in 2× loading buffer for 5 min, the supernatant was subjected to Western blotting analysis. ( B ) p53 ubiquitination was decreased after butein treatment. After incubation with butein for 24 h, HepG2 cells were treated with MG132 for another 6 h, the cell lysate was immunoprecipitated with p53 antibody and p53 ubiquitination was examined with anti-ubiquitin antibody. Abbreviations: IP, immunoprecipitation; WCE, whole cell extract.

Article Snippet: The p53 firefly luciferase reporter plasmid (pp53-TA-luc; Beyotime Biotechnology) and the internal control Renilla luciferase reporter plasmid (Promega) were cotransfected into HepG2 using Lipofectamine 2000 according to supplier’s directions.

Techniques: Ubiquitin Proteomics, Binding Assay, Co-Immunoprecipitation Assay, Incubation, Western Blot, Immunoprecipitation

Journal: OncoTargets and therapy

Article Title: Butein activates p53 in hepatocellular carcinoma cells via blocking MDM2-mediated ubiquitination

doi: 10.2147/OTT.S160119

Figure Lengend Snippet: Silencing of p53 impaired the sensitivity to butein. Notes: HepG2 cells were transfected with GFP shRNA or p53 shRNA, respectively, and then treated with 60 μM butein for 24 h. ( A ) The expressions of indicated proteins were examined by Western blotting. ( B ) The cells were stained with Annexin V-FITC and PI and analyzed with flow cytometry. * p <0.05, versus the sh-GFP group. Abbreviation: PI, propidium iodide.

Article Snippet: The p53 firefly luciferase reporter plasmid (pp53-TA-luc; Beyotime Biotechnology) and the internal control Renilla luciferase reporter plasmid (Promega) were cotransfected into HepG2 using Lipofectamine 2000 according to supplier’s directions.

Techniques: Transfection, shRNA, Western Blot, Staining, Flow Cytometry

Journal: OncoTargets and therapy

Article Title: Butein activates p53 in hepatocellular carcinoma cells via blocking MDM2-mediated ubiquitination

doi: 10.2147/OTT.S160119

Figure Lengend Snippet: Butein inhibited HepG2 xenograft growth in vivo. Notes: ( A ) The tumor growth curve. ( B ) The photograph of the tumors. ( C ) The tumor weights in the vehicle- and the butein-treated group, * p <0.05 versus the vehicle. Nude mice with HepG2 xenograft were randomly assigned into the vehicle or the treatment group, and the treatment was intraperitoneally injected (5 mg/kg butein) three times per week; the tumor volume was recorded twice per week. ( D ) Body weight of tumor-bearing mice in vehicle- and butein-treated group. ( E ) The expression of p53 and Ki67 in tumor tissue. The tumor tissue of HepG2 xenograft model was stained through immunohistochemistry with the indicated antibodies. Left: representative images of immunohistochemistry staining. Right: quantification of indicated marker. * p <0.05 indicated significant difference.

Article Snippet: The p53 firefly luciferase reporter plasmid (pp53-TA-luc; Beyotime Biotechnology) and the internal control Renilla luciferase reporter plasmid (Promega) were cotransfected into HepG2 using Lipofectamine 2000 according to supplier’s directions.

Techniques: In Vivo, Injection, Expressing, Staining, Immunohistochemistry, Marker

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: Dual-luciferase reporter gene assay 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: Dual-luciferase reporter gene assay 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: Dual-luciferase reporter gene assay 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: 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: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-04950-1

Figure Lengend Snippet: p53 inactivation causes the failure of senescence induction in Wwox −/− MEFs. a The upper panel is the schematic diagram of p53 protein. The amino acid sequence alignment of human and mouse p53 DBD is shown in the lower panel. TAD, transactivation domain; PRD, proline-rich domain; DBD, DNA-binding domain; TD, tetramerization domain; CRD, C-terminal regulatory domain. b , c The sequencing results of p53 gene coding region from Wwox +/− (clone no. 54) and Wwox −/− (clone no. 56) MEFs at passages 1 (early), 8 (intermediate) and 20 (late) are shown. In b , the nucleotide 638 from the translational start site of murine p53 changed from thymine to guanine, leading to a substitution of valine to glycine at position 213 in the intermediate and late-passage Wwox −/− MEF clone no. 56. In c , the change of nucleotide 403 from guanine to cytosine resulted in a substitution of alanine to proline in p53 in the late-passage Wwox −/− MEF clone no. 56. d Subcellular protein fractionation and western blotting were performed to examine the nuclear and cytosolic expression of p53 and p21 Cip1/Waf1 in late-passage Wwox +/− and Wwox −/− MEFs (clone no. 54 and 56, respectively). Histone H3 and GAPDH were used as nuclear and cytosolic protein controls, respectively. e ChIP assay was conducted using the chromatin isolated from late-passage Wwox +/− and Wwox −/− MEFs (clone no. 54 and 56, respectively). The immunoprecipitated chromatin using a control or anti-p53 antibody was analyzed for the presence of p21 Cip1/Waf1 promoter sequence (from − 200 to 98) by real-time PCR using the specific primers shown in Supplementary Table 1. The quantitative results of ChIP assay were normalized by the input control groups. Data are presented as mean ± SD from three independent experiments. *** P ≤ 0.005; Two-tailed paired t test. f A reporter construct containing a p53-driven promoter sequence (pp53-TA-Luc) was transfected into MEFs. The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) was used as an internal control. Luciferase activities were determined at 16 h after transfection. Data are presented as mean ± SD from three independent experiments. *** P ≤ 0.005; Two-tailed t test. g The protein expression of endogenous p21 Cip1/Waf1 (black arrow), p53 and WWOX and ectopic GFP-tagged human p53 (GFP-p53; black arrowhead) in late-passage Wwox +/+ , Wwox +/− , Wwox −/− MEFs (clone no. 32-5, 54 and 56, respectively) was examined by western blotting. β-actin was used as an internal control. M.W., molecular weight. h Cell growth rates of late-passage Wwox +/+ , Wwox +/− , Wwox −/− (clone no. 32-5, 54 and 56, respectively) and Wwox −/− MEFs expressing ectopic GFP-p53 or GFP protein. N.S. not significant; *** P ≤ 0.005; Two-tailed t test. i SA-β-gal staining of late-passage Wwox +/+ , Wwox +/− , Wwox −/− (clone no. 32-5, 54 and 56, respectively) and Wwox −/− MEFs expressing ectopic GFP-p53 protein. The lower panel pictures are the magnified images from the blue boxed areas in the upper panel. The percentages of SA-β-gal-positive senescent cells (red arrowheads) are shown at the right panel. Scale bars = 100 µm. * P ≤ 0.05; *** P ≤ 0.005; Two-tailed t test

Article Snippet: The pp53-TA-Luc luciferase-based reporter plasmid harboring a p53 response element (#PT3511-5W; Clontech, Mountain View, CA, USA) was used for monitoring p53-mediated signaling in MEFs.

Techniques: Sequencing, Binding Assay, Fractionation, Western Blot, Expressing, Isolation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test, Construct, Transfection, Luciferase, Plasmid Preparation, Molecular Weight, Staining

Journal:

Article Title: A global suppressor motif for p53 cancer mutants

doi: 10.1073/pnas.0401162101

Figure Lengend Snippet: The effect of suppressor amino acid changes on p53 cancer mutants in the p53 yeast assay. (A) PCR-mediated mutagenesis yielded suppressor mutation combinations that rescued transcriptional activity of four of the eight most common p53 cancer mutants, G245S, R249S, R273C, and R273H. SC-His plates select for the p53 expression plasmids and SC-Ura plates select for transcriptionally active p53. Pictures were taken after 2 days of incubation at 30°C. All yeast clones are represented by two patches. p53 cancer mutants are named (e.g., G245S), p53 mutants with cancer and suppressor combinations are numbered, and are crossreferenced with Table 1. (B) Design of the oligonucleotide-based mutagenesis. Expression plasmids for 30 of the most common p53 cancer mutants were gapped by using PflMI and NsiI. Pairs of annealed oligonucleotides representing all amino acid changes at codons 239 or 240 were cloned into the gap. The design also included background mutagenesis of the remaining codons between 225 and 241 that resulted in approximately one misincorporation per 100 nucleotides. (C) The global suppressor motif 235-239-240 rescues 16 of 30 of the most common p53 cancer mutants in the yeast p53 assay. All yeast transformants were processed and are shown as described for A. V272M was Ura+, but less so than V272M plus N239W. Identification of V272M plus N239W was likely possible, because the selection for rescued p53 cancer mutants (plating of transformants directly onto SC-His-Ura plates) was more stringent than subsequent confirmation of phenotypes for yeast patches by replica plating. V272M was transcriptionally inactive in mammalian reporter gene assays, whereas V272M plus N239W showed transcriptional activity (see Figs. ​Figs.22 and ​and3).3). p53 mutants with cancer and suppressor combinations are numbered, and are crossreferenced with Table 2.

Article Snippet: Reporter gene plasmids with single p53 DBSs were based on pp53-TA-Luc (Clontech; ref 20 ).

Techniques: Mutagenesis, Activity Assay, Expressing, Incubation, Clone Assay, Selection

Journal:

Article Title: A global suppressor motif for p53 cancer mutants

doi: 10.1073/pnas.0401162101

Figure Lengend Snippet: The global 235-239-240 suppressor motif rescues many common p53 cancer mutants in mammalian reporter gene assays for single p53 DBSs. The transcriptional activity of p53 cancer mutants with and without suppressor amino acids was evaluated in transient reporter gene assays in p53-negative H1299 cells. Luciferase activity, adjusted for transfection efficiency by using Renilla luciferase activity, was determined 24 h after transfection. The adjusted luciferase activity of cell lysates transfected with reporter plasmids and WT p53 expression plasmid was set as 100%. Shown are the mean and SD for three independent experiments. The reporter gene plasmids contained the DBSs of the p53 target genes GML (gray bars) or KILLER/DR5 (black bars) in the context of a heterologous yeast promoter. p53 cancer mutants are written out, and p53 mutants with cancer and suppressor amino acids are indicated by numbers that can be crossreferenced with Table 2 and Fig. 1C. The majority of p53 cancer mutants was rescued by at least one suppressor combination with transcriptional activities ranging from 40% to 130% of WT p53 activity. In the case of R158L, V173M, Y205C, and Y220C, the presence of suppressor amino acids resulted in only 20% of WT p53 activity. However, this result still represents a significant rescue effect, considering the lack of activity of the p53 cancer mutants alone.

Article Snippet: Reporter gene plasmids with single p53 DBSs were based on pp53-TA-Luc (Clontech; ref 20 ).

Techniques: Activity Assay, Luciferase, Transfection, Expressing, Plasmid Preparation