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Addgene inc p53
P53, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcdna3+p53+wt/pcDNA3+p53+WT+(Plasmid+%2369003)/10__3390_slash_biom16020240-86-12-14
Average 93 stars, based on 43 article reviews

p53 - by Bioz Stars, 2026-10

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Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs
Article Snippet: For the overexpression of hnRNP E1, plasmid pT7-V5-SBP-C1-HshnRNP E1 was used which was a gift from Elisa Izaurralde (Addgene plasmid # 64921; http://n2t.net/addgene : 64921; RRID: Addgene_64921) and upon transfection, resulted in robust hnRNPE1 expression in the RNA levels (viz C , G , E , D , B , D , F , etc) ultimately leading to modest ectopic expression at the protein levels (viz , D and H , etc). .. For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003). ..

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3' UTR C-rich motifs.
Article Snippet: For the overexpression of hnRNP E1, plasmid pT7-V5-SBP-C1-HshnRNP E1 was used which was a gift from Elisa Izaurralde (Addgene plasmid # 64921; http://n2t.net/addgene: 64921; RRID: Addgene_64921), and upon transfection, resulted in robust hnRNPE1 expression in the RNA levels (viz Figs. 1C, 1G, 6E, 7D, S1B, D, F, etc) ultimately leading to modest ectopic expression at the protein levels (viz Fig. 1D, 1H, etc). .. For wild-type p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene: 69003; RRID: Addgene_69003). ..

Plasmid Preparation:

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hnRNP E1 silencing reduces p53 and p21 proteins but hnRNP E1 overexpression enhances p53 and p21 expression . A , bar graph showing the effect of siRNA-mediated silencing of hnRNP E1 on p53 and p21 mRNA levels in HepG2 cells, quantified by real-time PCR (RT-PCR). B , Western blot panels showing the effect of hnRNP E1 silencing on p53 and p21 protein levels in HepG2, MCF-7, A-549, and HEK-293 cells. Bar graph ( right ) shows normalized blot band intensities for hnRNP E1, p53, and p21. C , bar graph showing the effect of hnRNP E1 overexpression on p53 and p21 mRNA levels in HepG2 cells using RT-PCR. D , Western blot panels showing the effect of hnRNP E1 overexpression on the p53 and p21 protein levels in HepG2, MCF-7, A-549, and HEK-293 cells. Bar graph ( right ) represents normalized blot band intensities of hnRNP E1, p53, and p21. E , bar graph showing the effect of hnRNP E1 silencing on p53 and p21 mRNA levels in HuH7 cells using RT-PCR. F , Western blot panels showing the effect of hnRNP E1 silencing on p53 and p21 protein levels in Huh7 and MDA-MB-231 cells. Bar graph ( right ) shows normalized hnRNP E1, p53, and p21 band intensities. G , bar graph showing the effect of hnRNP E1 overexpression on p53 and p21 mRNA levels in Huh7 cells using RT-PCR. H , Western blot panels depicting the effect of hnRNP E1 overexpression on the p53 and p21 protein levels in Huh7 and MDA-MB-231 cells. Bar graph ( right ) shows the normalized band intensities of hnRNP E1, p53, and p21. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Experiments in A , C , E , and G were replicated thrice, and those in B , D , F , and H were repeated twice across cell types. hnRNP, heterogeneous nuclear ribonucleoprotein.

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 silencing reduces p53 and p21 proteins but hnRNP E1 overexpression enhances p53 and p21 expression . A , bar graph showing the effect of siRNA-mediated silencing of hnRNP E1 on p53 and p21 mRNA levels in HepG2 cells, quantified by real-time PCR (RT-PCR). B , Western blot panels showing the effect of hnRNP E1 silencing on p53 and p21 protein levels in HepG2, MCF-7, A-549, and HEK-293 cells. Bar graph ( right ) shows normalized blot band intensities for hnRNP E1, p53, and p21. C , bar graph showing the effect of hnRNP E1 overexpression on p53 and p21 mRNA levels in HepG2 cells using RT-PCR. D , Western blot panels showing the effect of hnRNP E1 overexpression on the p53 and p21 protein levels in HepG2, MCF-7, A-549, and HEK-293 cells. Bar graph ( right ) represents normalized blot band intensities of hnRNP E1, p53, and p21. E , bar graph showing the effect of hnRNP E1 silencing on p53 and p21 mRNA levels in HuH7 cells using RT-PCR. F , Western blot panels showing the effect of hnRNP E1 silencing on p53 and p21 protein levels in Huh7 and MDA-MB-231 cells. Bar graph ( right ) shows normalized hnRNP E1, p53, and p21 band intensities. G , bar graph showing the effect of hnRNP E1 overexpression on p53 and p21 mRNA levels in Huh7 cells using RT-PCR. H , Western blot panels depicting the effect of hnRNP E1 overexpression on the p53 and p21 protein levels in Huh7 and MDA-MB-231 cells. Bar graph ( right ) shows the normalized band intensities of hnRNP E1, p53, and p21. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Experiments in A , C , E , and G were replicated thrice, and those in B , D , F , and H were repeated twice across cell types. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

hnRNP E1 interacts with 3′-UTRs of p53 and p21 RNAs and enhances their stability . A – C , RNA–protein interaction heat maps generated using catRAPID, showing binding intensities of hnRNP E1 to the 3′-UTRs of p53 and p21; x -axis represents RNA nucleotide positions, and y -axis shows hnRNP E1 amino acid positions. Due to the large size of the p21 3′-UTR, the sequence was divided into proximal and distal segments for analysis. D , silver-stained SDS-PAGE gel showing the efficiency and specificity of RNA immunoprecipitation (RIP) using control (IgG) and V5 tag–based hnRNP E1 pulldown. E , agarose gel showing PCR-based confirmation of hnRNP E1 interactions with p53 and p21 RNAs in the RIP experiment. Dashed red boxes indicate the specific PCR-amplified products for p53 and p21. F and G , typhoon-scanned polyacrylamide gels from photoaffinity crosslinking experiment, showing direct interactions between 3′-UTRs of p53 and p21 with GST-hnRNP E1 fusion protein. Bands corresponding to the protein–RNA complexes are indicated with ∗. Lane 1 represents the blank control. No interaction was observed between the 5′-UTRs of either p53 or p21 with purified GST-hnRNP E1 fusion protein. H and I , mRNA decay assay for p53 and p21 under hnRNP E1-silenced conditions in HepG2 cells using actinomycin D, showing reduced mRNA stability upon hnRNP E1 depletion. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data ( F – I ) originated from three independent experiments. hnRNP, heterogeneous nuclear ribonucleoprotein.

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 interacts with 3′-UTRs of p53 and p21 RNAs and enhances their stability . A – C , RNA–protein interaction heat maps generated using catRAPID, showing binding intensities of hnRNP E1 to the 3′-UTRs of p53 and p21; x -axis represents RNA nucleotide positions, and y -axis shows hnRNP E1 amino acid positions. Due to the large size of the p21 3′-UTR, the sequence was divided into proximal and distal segments for analysis. D , silver-stained SDS-PAGE gel showing the efficiency and specificity of RNA immunoprecipitation (RIP) using control (IgG) and V5 tag–based hnRNP E1 pulldown. E , agarose gel showing PCR-based confirmation of hnRNP E1 interactions with p53 and p21 RNAs in the RIP experiment. Dashed red boxes indicate the specific PCR-amplified products for p53 and p21. F and G , typhoon-scanned polyacrylamide gels from photoaffinity crosslinking experiment, showing direct interactions between 3′-UTRs of p53 and p21 with GST-hnRNP E1 fusion protein. Bands corresponding to the protein–RNA complexes are indicated with ∗. Lane 1 represents the blank control. No interaction was observed between the 5′-UTRs of either p53 or p21 with purified GST-hnRNP E1 fusion protein. H and I , mRNA decay assay for p53 and p21 under hnRNP E1-silenced conditions in HepG2 cells using actinomycin D, showing reduced mRNA stability upon hnRNP E1 depletion. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data ( F – I ) originated from three independent experiments. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Generated, Binding Assay, Sequencing, Staining, SDS Page, RNA Immunoprecipitation, Control, Agarose Gel Electrophoresis, Amplification, Purification, Mrna Decay Assay

hnRNP E1 binds to C-rich RNA motifs on 3′-UTRs of p53 and p21 and modulates their regulation . A and B , predicted hnRNP E1 binding sites on p53 and p21 3′-UTRs were identified using catRAPID and are illustrated in schematic models of the respective mRNAs. C and D , typhoon-scanned polyacrylamide gels from photoaffinity crosslinking experiments show interactions between GST-hnRNP E1 fusion protein and WT 3′-UTRs of both p53 and p21. Bands corresponding to the interacting protein–RNA complexes are indicated with ∗. Motif-deleted mutant 3′-UTRs [3′-UTR(CY5) Mut; p53 Δ-motif 1/2/3/4 and p21 Δ-motif 1/2/3] showed no interaction (last lanes, both the gels). Lane 1 represents a blank control. No interaction of GST was observed with the 3′-UTRs of either p53 or p21. E and F , bar graph showing the functional role of each identified motif in the context of 3′-UTR–guided functional regulation of p53 and p21, by RT-PCR–based luciferase RNA levels and luciferase assays. HepG2 cell were transfected with either p53 or p21 5′-UTR-luciferase-3′-UTR expression plasmids or motif-deleted variants under hnRNP E1-silenced conditions (sihnRNP E1-mediated knockdown). Luciferase RNA levels and luciferase activity from siControl-transfected cells was normalized to 1, and relative luciferase RNA level and activity of WT and MUT (motif-deleted variants) 3′-UTRs is shown. The ratio of the normalized luciferase values to the normalized luciferase RNA levels are shown in the boxes below the bar graph. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Data represents findings from three independent experimental replicates. hnRNP, heterogeneous nuclear ribonucleoprotein.

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 binds to C-rich RNA motifs on 3′-UTRs of p53 and p21 and modulates their regulation . A and B , predicted hnRNP E1 binding sites on p53 and p21 3′-UTRs were identified using catRAPID and are illustrated in schematic models of the respective mRNAs. C and D , typhoon-scanned polyacrylamide gels from photoaffinity crosslinking experiments show interactions between GST-hnRNP E1 fusion protein and WT 3′-UTRs of both p53 and p21. Bands corresponding to the interacting protein–RNA complexes are indicated with ∗. Motif-deleted mutant 3′-UTRs [3′-UTR(CY5) Mut; p53 Δ-motif 1/2/3/4 and p21 Δ-motif 1/2/3] showed no interaction (last lanes, both the gels). Lane 1 represents a blank control. No interaction of GST was observed with the 3′-UTRs of either p53 or p21. E and F , bar graph showing the functional role of each identified motif in the context of 3′-UTR–guided functional regulation of p53 and p21, by RT-PCR–based luciferase RNA levels and luciferase assays. HepG2 cell were transfected with either p53 or p21 5′-UTR-luciferase-3′-UTR expression plasmids or motif-deleted variants under hnRNP E1-silenced conditions (sihnRNP E1-mediated knockdown). Luciferase RNA levels and luciferase activity from siControl-transfected cells was normalized to 1, and relative luciferase RNA level and activity of WT and MUT (motif-deleted variants) 3′-UTRs is shown. The ratio of the normalized luciferase values to the normalized luciferase RNA levels are shown in the boxes below the bar graph. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Data represents findings from three independent experimental replicates. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Binding Assay, Mutagenesis, Control, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Transfection, Expressing, Knockdown, Activity Assay

hnRNP E1 induces translation of p53 and p21 mRNA via their 3′-UTRs . A and B , bar graph showing the normalized (by setting control to 1) luciferase RNA levels using RT-PCR and luciferase assay–based translational efficiency under hnRNP E1-silenced or hnRNP E1-overexpressed cellular conditions in HepG2 cells transfected with p53 5′-UTR-luciferase-3′-UTR expression plasmid. C and D , bar graph showing the normalized (by setting control to 1) luciferase RNA levels using RT-PCR and luciferase assay–based translational efficiency under hnRNP E1-silenced or hnRNP E1-overexpressed cellular conditions in HepG2 cells transfected with p21 5′-UTR-luciferase-3′-UTR expression plasmid. Values in the boxes below the bar graph (A to D) indicate the ratio of normalized luciferase activity to luciferase RNA. E and F , line graph showing the in vitro translation assays of in vitro runoff transcripts containing both the 5′- and 3′-UTRs of p53 and p21 in the presence of increasing concentrations of purified GST-hnRNP E1 (GST-E1), showing time-dependent luciferase activity. G and H , similar to the experiment E and F, the in vitro translation assay for in vitro runoff transcripts containing only 5′-UTRs of p53 and p21. I and J , similar to the experiment E and F, the in vitro translation assay for in vitro runoff transcripts containing only 3′-UTRs of p53 and p21. All the experimental time points (5, 15, 30, 60, and 90 min) are presented in a linear scale. Schematic representations of transcript configurations (E to J) are provided. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Experiments were performed in triplicate. hnRNP, heterogeneous nuclear ribonucleoprotein.

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 induces translation of p53 and p21 mRNA via their 3′-UTRs . A and B , bar graph showing the normalized (by setting control to 1) luciferase RNA levels using RT-PCR and luciferase assay–based translational efficiency under hnRNP E1-silenced or hnRNP E1-overexpressed cellular conditions in HepG2 cells transfected with p53 5′-UTR-luciferase-3′-UTR expression plasmid. C and D , bar graph showing the normalized (by setting control to 1) luciferase RNA levels using RT-PCR and luciferase assay–based translational efficiency under hnRNP E1-silenced or hnRNP E1-overexpressed cellular conditions in HepG2 cells transfected with p21 5′-UTR-luciferase-3′-UTR expression plasmid. Values in the boxes below the bar graph (A to D) indicate the ratio of normalized luciferase activity to luciferase RNA. E and F , line graph showing the in vitro translation assays of in vitro runoff transcripts containing both the 5′- and 3′-UTRs of p53 and p21 in the presence of increasing concentrations of purified GST-hnRNP E1 (GST-E1), showing time-dependent luciferase activity. G and H , similar to the experiment E and F, the in vitro translation assay for in vitro runoff transcripts containing only 5′-UTRs of p53 and p21. I and J , similar to the experiment E and F, the in vitro translation assay for in vitro runoff transcripts containing only 3′-UTRs of p53 and p21. All the experimental time points (5, 15, 30, 60, and 90 min) are presented in a linear scale. Schematic representations of transcript configurations (E to J) are provided. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Experiments were performed in triplicate. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Control, Luciferase, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Activity Assay, In Vitro, Purification

$hnRNP E1 induces p53 and p21 mRNA polyribosome loading . A and B , ribosomal profiles from sucrose density gradient fractionation from HepG2 cells overexpressing or silenced for hnRNP E1. Fractionation efficiency demonstrated by probing for RPS6 and RPL11 distributions across alternative fractions with western blots. The hnRNP E1 silencing and overexpression are shown in the Western blot panels ( top right of each subfigure). C and D , semiquantitative midphase PCR showing the relative abundance of p53, p21, and β-actin (control) mRNAs, in the collected alternative fractions from polyribosome fractionation assay following RNA extraction and cDNA synthesis. E and F , line graphs showing the relative band intensities of the p53, p21, and β-actin cDNA amplicons under hnRNP E1–overexpressed and hnRNP E1-silenced conditions relative to the control (normalized to 1). Data originated from three independent experiments. hnRNP, heterogeneous nuclear ribonucleoprotein. cDNA, complementary DNA.$

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 induces p53 and p21 mRNA polyribosome loading . A and B , ribosomal profiles from sucrose density gradient fractionation from HepG2 cells overexpressing or silenced for hnRNP E1. Fractionation efficiency demonstrated by probing for RPS6 and RPL11 distributions across alternative fractions with western blots. The hnRNP E1 silencing and overexpression are shown in the Western blot panels ( top right of each subfigure). C and D , semiquantitative midphase PCR showing the relative abundance of p53, p21, and β-actin (control) mRNAs, in the collected alternative fractions from polyribosome fractionation assay following RNA extraction and cDNA synthesis. E and F , line graphs showing the relative band intensities of the p53, p21, and β-actin cDNA amplicons under hnRNP E1–overexpressed and hnRNP E1-silenced conditions relative to the control (normalized to 1). Data originated from three independent experiments. hnRNP, heterogeneous nuclear ribonucleoprotein. cDNA, complementary DNA.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Fractionation, Western Blot, Over Expression, Control, RNA Extraction, cDNA Synthesis

KH1 and KH2 domains of hnRNP E1 mediates p53/p21 expression . A , secondary structures of the KH domains of hnRNP E1 is shown. Dot matrix alignment of the three consensus KH domains (accession numbers NP_006187.2 ; CAA55016.1 ; NM_006196 ; BC039742 and X78137 ) is shown, where matches are shown as dots and the deletions/gaps as dashes . For positions of three α-helices ( blue ) and three β-sheets ( red ) of each KH domain are indicated with dashed boxes on the aligned sequences. B , sequence identity matrix showing the degree of sequence similarity among the three consensus KH domains of hnRNP E1. Matrix was generated using BioEdit software (version 7.2; https://bioedit.software.informer.com/7.2/#google_vignette ) after excluding the indel sites. C , pictorial map depicting the location of three KH domains of hnRNP E1 and different KH-deleted variants of the hnRNP E1 expression system with V5 and SBP tags. D , intracellular localization of full-length hnRNP E1 and KH domain–deleted variants detected by immunofluorescence assay. The scale bar represents 10 μm. E , bar graph showing the relative expression of p53 and p21 RNA in cellular conditions overexpressing full-length hnRNP E1 or its different KH-deleted variants, measured by realtime PCR assay. F , Western blot analysis of p53 and p21 protein levels under overexpression of full-length hnRNP E1 or its different KH-deleted variants. Bar graph ( right ) shows normalized protein band intensities of p53 and p21. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. The experiments ( D to F ) were replicated thrice. hnRNP, heterogeneous nuclear ribonucleoprotein.

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: KH1 and KH2 domains of hnRNP E1 mediates p53/p21 expression . A , secondary structures of the KH domains of hnRNP E1 is shown. Dot matrix alignment of the three consensus KH domains (accession numbers NP_006187.2 ; CAA55016.1 ; NM_006196 ; BC039742 and X78137 ) is shown, where matches are shown as dots and the deletions/gaps as dashes . For positions of three α-helices ( blue ) and three β-sheets ( red ) of each KH domain are indicated with dashed boxes on the aligned sequences. B , sequence identity matrix showing the degree of sequence similarity among the three consensus KH domains of hnRNP E1. Matrix was generated using BioEdit software (version 7.2; https://bioedit.software.informer.com/7.2/#google_vignette ) after excluding the indel sites. C , pictorial map depicting the location of three KH domains of hnRNP E1 and different KH-deleted variants of the hnRNP E1 expression system with V5 and SBP tags. D , intracellular localization of full-length hnRNP E1 and KH domain–deleted variants detected by immunofluorescence assay. The scale bar represents 10 μm. E , bar graph showing the relative expression of p53 and p21 RNA in cellular conditions overexpressing full-length hnRNP E1 or its different KH-deleted variants, measured by realtime PCR assay. F , Western blot analysis of p53 and p21 protein levels under overexpression of full-length hnRNP E1 or its different KH-deleted variants. Bar graph ( right ) shows normalized protein band intensities of p53 and p21. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. The experiments ( D to F ) were replicated thrice. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Expressing, Sequencing, Generated, Software, Immunofluorescence, Western Blot, Over Expression

$KH1 and KH2 domains of hnRNP E1 independently binds to 3′-UTRs of p53/p21 and modulate their translation . A , different KH domains with V5 and SBP tags are shown in the figure. B , Western blots showing nuclear versus cytoplasmic distribution of full-length hnRNP E1 and its KH domains based on fractionation assays. C , intracellular localization of hnRNP E1 and its KH domains detected by immunofluorescence assay. The scale bar represents 10 μm. Bar graph ( bottom ) shows the relative cytoplasmic localization scores normalized to nuclear localization score (set to 1). D , bar graph showing the relative expression of p53 and p21 RNA in cellular conditions overexpressing full-length hnRNP E1 or its different KH domains measured by real-time PCR. E , Western blot analysis of p53 and p21 protein levels under overexpression of full-length hnRNP E1 or its KH domains. Bar graph ( right ) displays the corresponding normalized protein band intensities. F and G , bar graph showing the effect of different KH domains on the translational efficiency measured using luciferase assays. HepG2 cells were cotransfected with each KH domain–encoding plasmids and either p53 5′-UTR-luciferase-3′-UTR ( F ) or with p21 5′-UTR-luciferase-3′-UTR ( G ) expression plasmids. H and I , typhoon-scanned polyacrylamide gels of the photoaffinity crosslinking experiments showing interactions of 3′-UTRs of p53 and p21 with GST-KH fusion proteins. Bands corresponding to the proteins–RNA complexes are indicated with ∗. Lane 1, blank control; GST-hnRNP E1 (full-length), served as positive control. J and K , line graph showing the time-dependent in vitro translation efficiency (luciferase signal) of in vitro runoff transcripts containing both 5′- and 3′-UTRs of p53 and p21, respectively, in the presence of purified GST-hnRNP E1 (GST-E1) and GST-KH (1/2/3) fusion proteins at equal concentrations (100 nM). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. The experiments ( C – K ) were performed in triplicate. hnRNP, heterogeneous nuclear ribonucleoprotein.$

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: KH1 and KH2 domains of hnRNP E1 independently binds to 3′-UTRs of p53/p21 and modulate their translation . A , different KH domains with V5 and SBP tags are shown in the figure. B , Western blots showing nuclear versus cytoplasmic distribution of full-length hnRNP E1 and its KH domains based on fractionation assays. C , intracellular localization of hnRNP E1 and its KH domains detected by immunofluorescence assay. The scale bar represents 10 μm. Bar graph ( bottom ) shows the relative cytoplasmic localization scores normalized to nuclear localization score (set to 1). D , bar graph showing the relative expression of p53 and p21 RNA in cellular conditions overexpressing full-length hnRNP E1 or its different KH domains measured by real-time PCR. E , Western blot analysis of p53 and p21 protein levels under overexpression of full-length hnRNP E1 or its KH domains. Bar graph ( right ) displays the corresponding normalized protein band intensities. F and G , bar graph showing the effect of different KH domains on the translational efficiency measured using luciferase assays. HepG2 cells were cotransfected with each KH domain–encoding plasmids and either p53 5′-UTR-luciferase-3′-UTR ( F ) or with p21 5′-UTR-luciferase-3′-UTR ( G ) expression plasmids. H and I , typhoon-scanned polyacrylamide gels of the photoaffinity crosslinking experiments showing interactions of 3′-UTRs of p53 and p21 with GST-KH fusion proteins. Bands corresponding to the proteins–RNA complexes are indicated with ∗. Lane 1, blank control; GST-hnRNP E1 (full-length), served as positive control. J and K , line graph showing the time-dependent in vitro translation efficiency (luciferase signal) of in vitro runoff transcripts containing both 5′- and 3′-UTRs of p53 and p21, respectively, in the presence of purified GST-hnRNP E1 (GST-E1) and GST-KH (1/2/3) fusion proteins at equal concentrations (100 nM). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. The experiments ( C – K ) were performed in triplicate. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Western Blot, Fractionation, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Over Expression, Luciferase, Control, Positive Control, In Vitro, Purification

A ZNF-p53 fusion increases p53 SUMOylation with SUMO2. A , top : full ZNF451 protein, with the main domains identified. ZNF451 contains 2 SUMO-interacting motifs (SIMs), 12 C 2 H 2 Zinc Fingers, and a ubiquitin-interacting motif (UIM). Middle : the complete sequence of WT ZNF and ZNF AAAA tags used in this study. Bottom : pictograms illustrating p53 (in purple ) either not fused ( left ) or fused ( right ) with ZNF, the SUMO E3 module of WT ZNF451 ( green arrows ), and the SUMO-charged E2 (in gray ). B , E , in vitro assays comparing the SUMOylation of HA-p53, HA-p53+ZNF, and HA-ZNF-p53 ( B ) or of HA-p53, HA-ZNF-p53, and HA-ZNF AAAA -p53 ( E ) in the presence of E1 (SAE1 and SAE2), E2 (not visible on gel), and SUMO2. Reactions were started by adding 2 mM ATP and stopped at the indicated time points by the addition of Laemmli buffer. For ULP1-treated samples, reactions were allowed to proceed for 80 min, then 1 μM ULP1 was added for 10 min. Reactions were loaded on a 12% SDS-PAGE and colored with Coomassie blue . Stars represent SUMOylation states (∗ mono-SUMOylation, ∗∗ di-SUMOylation, ∗∗∗ tri-SUMOylation, and ∗∗∗∗ poly-SUMOylation). Gels are representative of three independent experiments (N = 3). C – F , relative abundance of SUMOylated and non-SUMOylated p53 determined by quantification of band intensities for the conditions presented in B or E . Detailed quantifications are shown on and , respectively. D , quantification of the intensity of the band corresponding to diSUMO2 forms of HA-ZNF-p53 and HA-p53+ZNF in B .

Journal: The Journal of Biological Chemistry

Article Title: A short SUMOylation tag modulates transcription factor activity

doi: 10.1016/j.jbc.2025.110807

Figure Lengend Snippet: A ZNF-p53 fusion increases p53 SUMOylation with SUMO2. A , top : full ZNF451 protein, with the main domains identified. ZNF451 contains 2 SUMO-interacting motifs (SIMs), 12 C 2 H 2 Zinc Fingers, and a ubiquitin-interacting motif (UIM). Middle : the complete sequence of WT ZNF and ZNF AAAA tags used in this study. Bottom : pictograms illustrating p53 (in purple ) either not fused ( left ) or fused ( right ) with ZNF, the SUMO E3 module of WT ZNF451 ( green arrows ), and the SUMO-charged E2 (in gray ). B , E , in vitro assays comparing the SUMOylation of HA-p53, HA-p53+ZNF, and HA-ZNF-p53 ( B ) or of HA-p53, HA-ZNF-p53, and HA-ZNF AAAA -p53 ( E ) in the presence of E1 (SAE1 and SAE2), E2 (not visible on gel), and SUMO2. Reactions were started by adding 2 mM ATP and stopped at the indicated time points by the addition of Laemmli buffer. For ULP1-treated samples, reactions were allowed to proceed for 80 min, then 1 μM ULP1 was added for 10 min. Reactions were loaded on a 12% SDS-PAGE and colored with Coomassie blue . Stars represent SUMOylation states (∗ mono-SUMOylation, ∗∗ di-SUMOylation, ∗∗∗ tri-SUMOylation, and ∗∗∗∗ poly-SUMOylation). Gels are representative of three independent experiments (N = 3). C – F , relative abundance of SUMOylated and non-SUMOylated p53 determined by quantification of band intensities for the conditions presented in B or E . Detailed quantifications are shown on and , respectively. D , quantification of the intensity of the band corresponding to diSUMO2 forms of HA-ZNF-p53 and HA-p53+ZNF in B .

Article Snippet: ZNF (ZNF451 24–55 ) was synthesized (Integrated DNA Technologies), while pcDNA3 p53 WT was a gift from David Meek (Addgene plasmid No. 69003; http://n2t.net/addgene:69003 ; RRID:Addgene_69003).

Techniques: Zinc-Fingers, Ubiquitin Proteomics, Sequencing, In Vitro, SDS Page

ZNF SUMO E3 ligase displays less activity with SUMO1 and promotes poly/multi SUMOylation of HA-ZNF-p53. A , C , and E , in vitro assays comparing the SUMOylation of A , HA-p53 in the presence of ZNF (HA-p53 + ZNF) and the HA-ZNF-p53 fusion using SUMO1, C , HA-ZNF-p53 using SUMO1 or SUMO2, and E , HA-ZNF-p53 or HA-p53+ZNF using SUMO2 K0. In all cases, HA-p53 was used as a control for basal SUMOylation. Reactions were performed as in . Representative Coomassie blue-stained 12% SDS-polyacrylamide gels from three independent experiments (N = 3) are shown. B , D , and F , relative abundance of SUMOylated and non-SUMOylated p53 determined by quantification of the band intensities for the conditions presented in A , C , and E , respectively. Detailed quantifications are shown in , and .

Journal: The Journal of Biological Chemistry

Article Title: A short SUMOylation tag modulates transcription factor activity

doi: 10.1016/j.jbc.2025.110807

Figure Lengend Snippet: ZNF SUMO E3 ligase displays less activity with SUMO1 and promotes poly/multi SUMOylation of HA-ZNF-p53. A , C , and E , in vitro assays comparing the SUMOylation of A , HA-p53 in the presence of ZNF (HA-p53 + ZNF) and the HA-ZNF-p53 fusion using SUMO1, C , HA-ZNF-p53 using SUMO1 or SUMO2, and E , HA-ZNF-p53 or HA-p53+ZNF using SUMO2 K0. In all cases, HA-p53 was used as a control for basal SUMOylation. Reactions were performed as in . Representative Coomassie blue-stained 12% SDS-polyacrylamide gels from three independent experiments (N = 3) are shown. B , D , and F , relative abundance of SUMOylated and non-SUMOylated p53 determined by quantification of the band intensities for the conditions presented in A , C , and E , respectively. Detailed quantifications are shown in , and .

Techniques: Activity Assay, In Vitro, Control, Staining

Mutation of the main SUMOylation acceptor site of HA-ZNF-p53 delays the monoSUMOylation of p53 but does not affect its poly/multi-SUMOylation. A , pictogram illustrating the mutation and sequence of p53 and p53 K386R . B – E , in vitro assays comparing the SUMOylation of HA-p53, HA-ZNF-p53, and HA-ZNF-p53 K386R in the presence of E1, E2, and SUMO2 ( B ) or SUMO2 K0 ( E ). Reactions were performed as in . Gels are representative of three independent experiments (N = 3). C – F , relative abundance of SUMOylated and non-SUMOylated p53 determined by quantification of the band intensities for the conditions presented in B – E . Detailed quantifications are shown on , and . D , quantification of the intensity of the band corresponding to diSUMO2 in HA-ZNF-p53 and HA-ZNF-p53 K386R conditions.

Journal: The Journal of Biological Chemistry

Article Title: A short SUMOylation tag modulates transcription factor activity

doi: 10.1016/j.jbc.2025.110807

Figure Lengend Snippet: Mutation of the main SUMOylation acceptor site of HA-ZNF-p53 delays the monoSUMOylation of p53 but does not affect its poly/multi-SUMOylation. A , pictogram illustrating the mutation and sequence of p53 and p53 K386R . B – E , in vitro assays comparing the SUMOylation of HA-p53, HA-ZNF-p53, and HA-ZNF-p53 K386R in the presence of E1, E2, and SUMO2 ( B ) or SUMO2 K0 ( E ). Reactions were performed as in . Gels are representative of three independent experiments (N = 3). C – F , relative abundance of SUMOylated and non-SUMOylated p53 determined by quantification of the band intensities for the conditions presented in B – E . Detailed quantifications are shown on , and . D , quantification of the intensity of the band corresponding to diSUMO2 in HA-ZNF-p53 and HA-ZNF-p53 K386R conditions.

Techniques: Mutagenesis, Sequencing, In Vitro

Fusion of ZNF to p53 increases its SUMOylation with SUMO2 but little with SUMO1 in HEK293 cells. A , immunoblot analysis of whole cell lysates or immunoprecipitated HA-p53, HA-ZNF-p53, HA-ZNF AAAA -p53, or HA-p53-SUMO2 following expression in HEK293 cells for 48 h. HEK293 cells were treated 4 h with ML792 or control (0.01% DMSO) then lysed. Immunoprecipitation assays were performed overnight at 4 °C using anti-HA antibodies, followed by washes and elution in Laemmli buffer. Proteins were separated on a 7.5% SDS-PAGE. Immunoblots were performed using anti-HA antibodies on whole cell lysates and anti-HA or anti-SUMO2/3 antibodies on immunoprecipitated proteins. B , relative SUMOylation levels for HA-ZNF-p53 and HA-ZNF AAAA -p53 were obtained from anti-HA and anti-SUMO2/3 immunoblots presented in A after normalization with HA-p53-SUMO2 for which association between p53 to SUMO2 is equimolar. C , immunoblot analysis of immunoprecipitated HA-p53, HA-ZNF-p53, HA-ZNF AAAA -p53, or HA-p53-SUMO1 following expression in HEK293 cells with detection using anti-HA or anti-SUMO1 antibodies. Representative immunoblots from three independent experiments (N = 3) are shown. D , identification of potentials SUMOylation sites in p53 using PhosphoSitePlus ( blue ), GPS-SUMO ( red ), and DeepSUMO ( green ). Confirmed (PhosphoSitePlus) or high-confidence (GPS-SUMO and DeepSUMO) predictions are represented with colored dots. E , mass spectrum of HA-ZNF-p53 transfected in HEK293 cells stably expressing His 6 -SUMO3 Q87R/Q88N and showing SUMOylation on lysine 386 and phosphorylation on Ser392. DMSO, dimethyl sulfoxide; HA, hemagglutinin.

Journal: The Journal of Biological Chemistry

Article Title: A short SUMOylation tag modulates transcription factor activity

doi: 10.1016/j.jbc.2025.110807

Figure Lengend Snippet: Fusion of ZNF to p53 increases its SUMOylation with SUMO2 but little with SUMO1 in HEK293 cells. A , immunoblot analysis of whole cell lysates or immunoprecipitated HA-p53, HA-ZNF-p53, HA-ZNF AAAA -p53, or HA-p53-SUMO2 following expression in HEK293 cells for 48 h. HEK293 cells were treated 4 h with ML792 or control (0.01% DMSO) then lysed. Immunoprecipitation assays were performed overnight at 4 °C using anti-HA antibodies, followed by washes and elution in Laemmli buffer. Proteins were separated on a 7.5% SDS-PAGE. Immunoblots were performed using anti-HA antibodies on whole cell lysates and anti-HA or anti-SUMO2/3 antibodies on immunoprecipitated proteins. B , relative SUMOylation levels for HA-ZNF-p53 and HA-ZNF AAAA -p53 were obtained from anti-HA and anti-SUMO2/3 immunoblots presented in A after normalization with HA-p53-SUMO2 for which association between p53 to SUMO2 is equimolar. C , immunoblot analysis of immunoprecipitated HA-p53, HA-ZNF-p53, HA-ZNF AAAA -p53, or HA-p53-SUMO1 following expression in HEK293 cells with detection using anti-HA or anti-SUMO1 antibodies. Representative immunoblots from three independent experiments (N = 3) are shown. D , identification of potentials SUMOylation sites in p53 using PhosphoSitePlus ( blue ), GPS-SUMO ( red ), and DeepSUMO ( green ). Confirmed (PhosphoSitePlus) or high-confidence (GPS-SUMO and DeepSUMO) predictions are represented with colored dots. E , mass spectrum of HA-ZNF-p53 transfected in HEK293 cells stably expressing His 6 -SUMO3 Q87R/Q88N and showing SUMOylation on lysine 386 and phosphorylation on Ser392. DMSO, dimethyl sulfoxide; HA, hemagglutinin.

Techniques: Western Blot, Immunoprecipitation, Expressing, Control, SDS Page, Transfection, Stable Transfection, Phospho-proteomics

ZNF-mediated SUMOylation of p53 does not promote strong degradation by the proteasome. A – C , immunoblot analysis of immunoprecipitated HA-p53, HA-ZNF-p53, or HA-ZNF AAAA -p53 from whole cell extracts of HEK293 cells transfected for 24 or 48 h ( A ), or with HA-p53 or HA-ZNF-p53 for 48 h followed by treatment with cycloheximide (CHX) or control (DMSO 0.01%) for 30 min, then with MG132 or control (DMSO 0.01%) for 4 h ( C ). Proteins were separated on a 7.5% SDS-PAGE and immunoblots were performed using anti-HA or anti-SUMO2/3 antibodies. Representative immunoblots from three independent experiments (N = 3) are shown. B – E , quantification of SUMOylation levels was performed by measuring the band intensities for each condition of immunoprecipitated proteins and is presented relative to the control treatment. D , relative abundance of HA-ZNF-p53 was obtained by dividing the band intensities for each condition in the whole cell lysate immunoblot by the total band intensity. Normality was assessed using the Shapiro-Wilk test. A one-way ANOVA statistical analysis, followed by Tukey’s multiple comparison test, was performed to compare transfection conditions within the same treatment (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001). For SUMOylation quantification, SUMOylated over non-SUMOylated protein ratios were obtained by dividing the relative band intensity measured in anti-SUMO2/3 immunoblot by the relative band intensity measured in the HA immunoblots. DMSO, dimethyl sulfoxide; HA, hemagglutinin.

Journal: The Journal of Biological Chemistry

Article Title: A short SUMOylation tag modulates transcription factor activity

doi: 10.1016/j.jbc.2025.110807

Figure Lengend Snippet: ZNF-mediated SUMOylation of p53 does not promote strong degradation by the proteasome. A – C , immunoblot analysis of immunoprecipitated HA-p53, HA-ZNF-p53, or HA-ZNF AAAA -p53 from whole cell extracts of HEK293 cells transfected for 24 or 48 h ( A ), or with HA-p53 or HA-ZNF-p53 for 48 h followed by treatment with cycloheximide (CHX) or control (DMSO 0.01%) for 30 min, then with MG132 or control (DMSO 0.01%) for 4 h ( C ). Proteins were separated on a 7.5% SDS-PAGE and immunoblots were performed using anti-HA or anti-SUMO2/3 antibodies. Representative immunoblots from three independent experiments (N = 3) are shown. B – E , quantification of SUMOylation levels was performed by measuring the band intensities for each condition of immunoprecipitated proteins and is presented relative to the control treatment. D , relative abundance of HA-ZNF-p53 was obtained by dividing the band intensities for each condition in the whole cell lysate immunoblot by the total band intensity. Normality was assessed using the Shapiro-Wilk test. A one-way ANOVA statistical analysis, followed by Tukey’s multiple comparison test, was performed to compare transfection conditions within the same treatment (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001). For SUMOylation quantification, SUMOylated over non-SUMOylated protein ratios were obtained by dividing the relative band intensity measured in anti-SUMO2/3 immunoblot by the relative band intensity measured in the HA immunoblots. DMSO, dimethyl sulfoxide; HA, hemagglutinin.

Techniques: Western Blot, Immunoprecipitation, Transfection, Control, SDS Page, Comparison

Targeted SUMOylation of p53 diminishes its transcriptional activity. A , C , and G , the transcriptional activities of ectopically expressed p53 fusion proteins were assessed using the p53 luciferase reporter vector pG13-luc. Luciferase activity was measured after HEK293 cell transfection for 48 h with indicated expression vectors for p53, ZNF-p53, ZNF AAAA -p53. ZNF 2SIMSAAAA -p53 or the parental empty vector (EV) and treatment with SUMOylation inhibitors ML792 or TAK981 (5 μM), the MDM2 inhibitor Nutlin3A (5 μM) or control (DMSO 0.05%) for the last 24 h. Normality was assessed using the Shapiro-Wilk test. A one-way ANOVA statistical analysis followed by Tukey’s multiple comparison test was performed to compare transfection conditions within the same treatment. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. Representative experiments from N = 3 independent experiments are shown. B , D , and H , protein expression was assessed by immunoblotting in the same extracts as in ( A , C , and G ). E , sequences of the 9 ZNF mutants generated F , immunoblot analysis of whole cell lysates HA-p53, HA-ZNF-p53, and the nine ZNF-fusion mutants following expression in HEK293 cells for 48 h. Cells were then lysed, and proteins were separated on a 7.5% SDS-PAGE, transferred to a membrane using a Trans-Blot Turbo system and immunoblots were performed using anti-HA antibody. The same amounts of transfected proteins were loaded on each gels. Representative immunoblots from two independent experiments (N = 2) are shown. DMSO, dimethyl sulfoxide.

Journal: The Journal of Biological Chemistry

Article Title: A short SUMOylation tag modulates transcription factor activity

doi: 10.1016/j.jbc.2025.110807

Figure Lengend Snippet: Targeted SUMOylation of p53 diminishes its transcriptional activity. A , C , and G , the transcriptional activities of ectopically expressed p53 fusion proteins were assessed using the p53 luciferase reporter vector pG13-luc. Luciferase activity was measured after HEK293 cell transfection for 48 h with indicated expression vectors for p53, ZNF-p53, ZNF AAAA -p53. ZNF 2SIMSAAAA -p53 or the parental empty vector (EV) and treatment with SUMOylation inhibitors ML792 or TAK981 (5 μM), the MDM2 inhibitor Nutlin3A (5 μM) or control (DMSO 0.05%) for the last 24 h. Normality was assessed using the Shapiro-Wilk test. A one-way ANOVA statistical analysis followed by Tukey’s multiple comparison test was performed to compare transfection conditions within the same treatment. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. Representative experiments from N = 3 independent experiments are shown. B , D , and H , protein expression was assessed by immunoblotting in the same extracts as in ( A , C , and G ). E , sequences of the 9 ZNF mutants generated F , immunoblot analysis of whole cell lysates HA-p53, HA-ZNF-p53, and the nine ZNF-fusion mutants following expression in HEK293 cells for 48 h. Cells were then lysed, and proteins were separated on a 7.5% SDS-PAGE, transferred to a membrane using a Trans-Blot Turbo system and immunoblots were performed using anti-HA antibody. The same amounts of transfected proteins were loaded on each gels. Representative immunoblots from two independent experiments (N = 2) are shown. DMSO, dimethyl sulfoxide.

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Transfection, Expressing, Control, Comparison, Western Blot, Generated, SDS Page, Membrane

Figure 1. Overexpression of TP53 variants and wild-type (WT) constructs selected for functional analysis. (A)—Distribution of TP53 variants analyzed in this study. TAD: transactivation domain, PRD: proline-rich domain, DBD: DNA-binding domain, NLS: nuclear localization signal, TD: tetramerization domain, and BD: basic domain. (B)—TP53 mRNA expression levels for all transfected constructs, including empty vector (EV), p53 wild-type (WT) and p53 variants. (C)—Analysis of transcriptional activation (TA) capacity of TP53 variants. Variant activity is represented in relation to wild-type construction activity. The black bars represent the positive (WT) and negative controls (EV), and the gray bars represent the analyzed variants. p-values < 0.05 were considered statistically significant; (*) p < 0.05; (****) p < 0.0001.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 1. Overexpression of TP53 variants and wild-type (WT) constructs selected for functional analysis. (A)—Distribution of TP53 variants analyzed in this study. TAD: transactivation domain, PRD: proline-rich domain, DBD: DNA-binding domain, NLS: nuclear localization signal, TD: tetramerization domain, and BD: basic domain. (B)—TP53 mRNA expression levels for all transfected constructs, including empty vector (EV), p53 wild-type (WT) and p53 variants. (C)—Analysis of transcriptional activation (TA) capacity of TP53 variants. Variant activity is represented in relation to wild-type construction activity. The black bars represent the positive (WT) and negative controls (EV), and the gray bars represent the analyzed variants. p-values < 0.05 were considered statistically significant; (*) p < 0.05; (****) p < 0.0001.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Over Expression, Construct, Functional Assay, Binding Assay, Expressing, Transfection, Plasmid Preparation, Activation Assay, Variant Assay, Activity Assay

Figure 2. p53 overexpression impact in mRNA relative expression of p53’s target genes. The quantifications were normalized by endogenous housekeeping gene GUSB. The WT conditions were settled = 1 and the mRNA expression of CDKN1A (A), MDM2 (B), GADD45A (C) and PMAIP (D) genes were relativized by WT. The statistical comparisons between the variants were performed with their respective WT conditions (control or 10 Gy). p-values < 0.05 (*) were considered statistically significant; (**) p < 0.005; (***) p < 0.001; and (****) p < 0.0001. UT = untreated.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 2. p53 overexpression impact in mRNA relative expression of p53’s target genes. The quantifications were normalized by endogenous housekeeping gene GUSB. The WT conditions were settled = 1 and the mRNA expression of CDKN1A (A), MDM2 (B), GADD45A (C) and PMAIP (D) genes were relativized by WT. The statistical comparisons between the variants were performed with their respective WT conditions (control or 10 Gy). p-values < 0.05 (*) were considered statistically significant; (**) p < 0.005; (***) p < 0.001; and (****) p < 0.0001. UT = untreated.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Over Expression, Expressing, Control

Figure 3. Analysis of p21 and p-p53 Ser15 protein levels upon DNA damage. (A)—Western blot of p53 (53KDa), p-p53 Ser15 (53KDa), p21 (21KDa) and β-actin (42KDa), in a control or irradiated (10 Gy) conditions. Cellular protein levels of p-p53Ser15 (B) and p21 (C) in SK-OV-3 cell line overexpressing exogenous p53 (WT or variants) were quantified in control and 4 h after ionizing irradiation (10 Gy) conditions. p-values < 0.05 (*) were considered statistically significant. The analyzes were performed from two independent experiments.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 3. Analysis of p21 and p-p53 Ser15 protein levels upon DNA damage. (A)—Western blot of p53 (53KDa), p-p53 Ser15 (53KDa), p21 (21KDa) and β-actin (42KDa), in a control or irradiated (10 Gy) conditions. Cellular protein levels of p-p53Ser15 (B) and p21 (C) in SK-OV-3 cell line overexpressing exogenous p53 (WT or variants) were quantified in control and 4 h after ionizing irradiation (10 Gy) conditions. p-values < 0.05 (*) were considered statistically significant. The analyzes were performed from two independent experiments.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Western Blot, Control, Irradiation

Figure 4. Immunofluorescence analysis of p53 protein levels in p53 WT and K139N or V197M variants after irradiation (10 Gy). (A)—Representative images showing nuclear localization of p53 in p53 WT and K139N or V197M variants; scale bar, 40 µm; (B)—The ratio between Integrated Density (IntDen) and cell number of each condition. The presence of variants decreased the nuclear p53 levels compared to p53 WT. p-values < 0.05 were considered statistically significant; (**) p < 0.005.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 4. Immunofluorescence analysis of p53 protein levels in p53 WT and K139N or V197M variants after irradiation (10 Gy). (A)—Representative images showing nuclear localization of p53 in p53 WT and K139N or V197M variants; scale bar, 40 µm; (B)—The ratio between Integrated Density (IntDen) and cell number of each condition. The presence of variants decreased the nuclear p53 levels compared to p53 WT. p-values < 0.05 were considered statistically significant; (**) p < 0.005.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Immunofluorescence, Irradiation

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