Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIPKIα interacts with MDM2 and regulates PIP 2 association. A and B , Co-IP of MDM2 with PIPKIα from MDA-MB-231 ( A ) and MDA-MB-468 cells ( B ) treated with 30 μM cisplatin or vehicle for 24 h. Data shown represent three independent experiments. The PIPKIα IB intensity was quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Veh, vehicle; Cis, cisplatin-treated; short, short-time exposure; long, long-time exposure. C , Co-IP of endogenous MDM2 from MDA-MB-231 cells treated with vehicle or 30 μM cisplatin for 24 h. The MDM2, PIPKIα, PIPKIγ, PIPKIIα, PIPKIIβ, p53, and GAPDH IPed by MDM2 were analyzed by IB. short, short-time exposure; long, long-time exposure. D , recombinant MDM2 protein (1 μg) was incubated with 0.125, 0.25, 0.5, 1, 2 or 4 μg of PIPKIα protein. MDM2 was pulled down, and the binding with PIPKIα was analyzed with an anti-PIPKIα antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. E , Quantification of nuclear PLA of PIPKIα-MDM2 in MDA-MB-231 cells treated with vehicle, cisplatin, or cisplatin plus siRNAs targeting PIPKIα for KD. For cisplatin treatment, 30 μM cisplatin was added for 24 h. PIPKIα KD was achieved using siRNAs for 24 h, followed by 24 h cisplatin treatment. n = 30 cells pooled from 3 independent experiments, 10 cells per experiment. Veh, vehicle; Cis, cisplatin-treated, KD; PIPKIα KD. F , MDA-MB-231 cells were transfected with siRNAs targeting PIPKIα. After 24 h of transfection, cells were treated with 30 μM cisplatin for 24 h. IB analyzed the expression of the indicated proteins, IBs were quantified, and the graph is shown as mean ± s.d. of n = 3 independent experiments. Cis, cisplatin-treated; KD, knockdown; Mock, empty vector.

Article Snippet: GST-tagged p53 6Q mutant was purified with GST Sepharose 4B (#17075604, Cytiva), while His-tagged MDM2, αBC, HSP27, and PIPKIα were purified with Ni-NTA-agarose (#166038887, Qiagen) as previously described ( ).

Techniques: Co-Immunoprecipitation Assay, Recombinant, Incubation, Binding Assay, Transfection, Expressing, Knockdown, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 regulates the interaction of MDM2, sHSPs and p53 and controls MDM2 stability. A and B , 0.1 μg of MDM2 recombinant protein and 0.1 μg of αBC ( A ) or HSP27 ( B ) protein were incubated with 0, 0.5, 1, 2, or 5 μM PIP 2 . MDM2 was pulled down, and the associated αBC or HSP27 was analyzed with an anti-αBC or an anti-HSP27 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-468 cells were transfected with siRNAs for αBC or HSP27 for 48 h. An empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB MDM2 IBs were quantified, and the graph is shown as mean ± s.d. of n = 4 independent experiments. KD, knockdown. D , MDA-MB-468 cells were transfected with siRNAs for αBC for 48 h. Before harvesting, cells were treated with 10 μM of MG132 for 4 h. Empty vector (Mock) was used as a negative control. Expression of the indicated proteins was analyzed by IB, and MDM2 IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. E , recombinant MDM2 protein (0.1 μg) and GST-tagged p53 WT or PIP2-binding-defective p53 6Q mutant (p53 6Q, 0.1 μg) were incubated with 0, 1, 5, 10, or 50 μM PIP 2 . MDM2 was pulled down, and the associated p53 was analyzed with an anti-p53 antibody. The graphs are shown as mean ± s.d. of n = 3 independent experiments. p53 WT, wild type p53; p53 6Q, p53 6Q mutant.

Article Snippet: GST-tagged p53 6Q mutant was purified with GST Sepharose 4B (#17075604, Cytiva), while His-tagged MDM2, αBC, HSP27, and PIPKIα were purified with Ni-NTA-agarose (#166038887, Qiagen) as previously described ( ).

Techniques: Recombinant, Incubation, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the MDM2-p53 interaction. A and B , 0.1 μg of MDM2 and GST-p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC ( A ) or HSP27 ( B ) in the presence or absence of 10 μM of PIP 2 . After pulling down with MDM2 antibody-conjugated beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. sup, supernatant; pel, pellet. C , 0.5 μg Bacteria (His-tagged) and mammalian-expressed (FLAG-tagged) MDM2 ( A ) and p53 ( B ) proteins were analyzed by fluorescence IB. Fluorescence IB detects purified proteins and PIP 2 stably associated to proteins. The MDM2 and PIP 2 association is shown in the merged image. D , recombinant MDM2 protein (0.1 μg) and FLAG-, His-p53, or GST-p53 6Q mutant proteins were incubated with 0, 1, 5, or 10 μM PIP 2 . MDM2 was pulled down, and the associated different p53 was analyzed with an anti-p53 antibody. The graphs are shown bound p53 normalized to the strongest signal (His-p53 with 10 μM PIP 2 ) as mean ± s.d. of n = 3 independent experiments. p53 6Q, p53 6Q mutant. E , 0.1 μg of FLAG-MDM2 and p53 recombinant protein were incubated with gradually increasing amounts (0, 250, 500 ng) of αBC or HSP27. After pulling down with MDM2 beads, the bound p53, αBC and HSP27 were analyzed with an anti-p53, anti-αBC or anti-HSP27 antibody in the supernatant and pellet. The graphs are shown as mean ± s.d. of n = 3 independent experiments. FLAG-tagged proteins; mammalian-derived proteins.

Article Snippet: GST-tagged p53 6Q mutant was purified with GST Sepharose 4B (#17075604, Cytiva), while His-tagged MDM2, αBC, HSP27, and PIPKIα were purified with Ni-NTA-agarose (#166038887, Qiagen) as previously described ( ).

Techniques: Recombinant, Incubation, Bacteria, Fluorescence, Purification, Stable Transfection, Mutagenesis, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the ubiquitination function of MDM2. A , for MDM2 autoubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM) for 1 h. Ubiquitin, MDM2, αBC and HSP27 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. B , for in vitro ubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of His- and FLAG-tagged MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with 1 μM of His- and FLAG-tagged p53 for 1 h. For IP, samples were incubated with anti-p53-conjugated agarose overnight. Ubiquitin, MDM2, and p53 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-231 cells were transfected with siRNAs for PIPKIα and treated with vehicle or 30 μM cisplatin for 24 h. Empty vector (Mock) was used as a negative control. After cells were treated with 10 μM of MG132 for 4 h, cells were harvested for IP of MDM2 and p53. Expression of the indicated proteins was analyzed by IB, and ubiquitin IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown; UT, untreated; Cis, cisplatin treated; Cis/MG132, Cisplatin/MG132 treated. D , MDA-MB-468 cells were transfected with siRNAs for αBC and HSP27 for 48 h in absence or presence of MG132. Empty vector (Mock) was used as a negative control. Cells were processed for IP of MDM2. IB was used to analyze indicated proteins, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown. E , a model of PIP 2 regulation of the interaction between MDM2 and sHSPs, which differentially control the stability of MDM2 and function of of MDM2. In the presence of PIPKIα, PIP 2 is linked to MDM2, which recruits αBC to MDM2 to stabilize it and enhances p53 binding. Conversely, in the absence of PIPKIα and PIP 2 , HSP27 is recruited to MDM2, which increases the ubiquitin E3 ligase activity of MDM2.

Article Snippet: GST-tagged p53 6Q mutant was purified with GST Sepharose 4B (#17075604, Cytiva), while His-tagged MDM2, αBC, HSP27, and PIPKIα were purified with Ni-NTA-agarose (#166038887, Qiagen) as previously described ( ).

Techniques: Ubiquitin Proteomics, Incubation, In Vitro, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Control, Binding Assay, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

Techniques: Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

doi: 10.1186/s12964-025-02640-y

Figure Lengend Snippet: Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation, Staining, Flow Cytometry

Journal: Asian Pacific Journal of Cancer Prevention

Article Title: The Association between p53 Expression and Histopathology Grade of Astrocytoma

doi: 10.31557/apjcp.2025.26.7.2521

Figure Lengend Snippet: Figure 1. Immunohistochemistry Staining of p53 Mutant (High Power View, Magnification 400x). (A). Intense nuclear staining in immunopositive in ³10% of tumor cells was interpreted as positive (p53 mutant). (B). Weak nuclear staining was interpreted as negative as immunonegative (p53 wild-type)

Article Snippet: The research samples comprised Formalin-Fixed Paraffin-Embedded (FFPE) for Hematoxylin-Eosin (HE) and immunohistochemical staining with Anti-p53 mutant (M00001-3, Boster, Pleasanton, CA, USA).

Techniques: Immunohistochemistry, Staining, Mutagenesis