gfp p53  (Addgene inc)

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: Purified VDAC1 directly interacts with purified p53 and reduces channel conductance of bilayer-reconstituted VDAC1. ( A ) Coomassie blue-stained SDS-PAGE profile of purified VDAC1 and p53 proteins. ( B , C ) MST measurements: purified VDAC1 (100 nM) ( B ) or p53 (2 μM) ( C ) were fluorescently labeled using a Nano Temper Protein-Labeling Kit BLUE and incubated 15 min with the indicated concentrations of p53 (1.14 nM–18.75 μM) or of VDAC1 (33.3 nM–5.33 μM), respectively. Then 3–5 μL of the samples were loaded into MST-grade glass capillaries, and thermophoresis was measured using a Monolith-NT115 apparatus. Data in B and C were analyzed using GraphPad Prism (version 10.6.1) to drive the binding constants (Kd) and Hill coefficients (nH). ( D ) Purified VDAC1 was reconstituted into a PLB and channel currents through it in response to voltage step 0–10 mV or 0–60 mV, recorded before and 5 min after the addition of p53 (0.5 μM). The dashed lines indicate zero current. ( E ) The effect of p53 on VDAC1 conductance as a function of voltage, from 60 mV to −60 mV. The average steady-state conductance at a given voltage (G) was normalized to the conductance at 10 mV (G0). The recordings were taken before (•) and 5 min after the addition of p53 (○) (n = 3). ( F ) Cells were subjected to in situ PLA to test for close association between VDAC1 (OMM) and p53 using specific antibodies. The ligation products appear in red; nuclei were DAPI-stained (blue). PLA using VDAC1 and matrix located citrate synthase (CS) are shown as negative control.

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Purification, Staining, SDS Page, Labeling, Incubation, Binding Assay, In Situ, Ligation, Negative Control

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: Silencing VDAC1 expression leads to p53 translocation to the nucleus. ( A ) HeLa cells were transfected to express p53-GFP 24 h post transfection. Cells were seeded on 35 mm coverslips cultured to a 60% confluency, stained with Mitotracker red (Invitrogen, Waltham, MS, USA), and imaged using a confocal microscope. Nuclei were stained with DAPI. Arrows point to the nuclear localization. ( B ) Control and si-NT- and si-(h)VDAC1-treated cells (100 nM si-NT or 75 or 100 nM of si-(h)VDAC1), 24 h post transfection cells were lysed and analyzed for VDAC1 expression level using immunoblotting and anti-VDAC1 antibodies. ( C ) Quantitative analysis of VDAC1 levels of the samples in ( B ). ( D , E ) p53 sub-cellular localization was analyzed in control cells transfected with 100 nM si-NT or 100 nM si-(h)VDAC1, and 24 h post-transfection, cells were fixed and stained with anti-p53 antibodies, followed by secondary Alexa Fluor 488-conjugated anti-rabbit antibodies (shown in green) and DAPI (blue), visualized with a confocal microscope ( D ), arrows point to the nuclear localization), and quantified ( E ). Results show means ± SEM (n = 3), ** p < 0.01, *** p < 0.001. ( F ) Nuclear and cytosolic fractions were prepared from si-NT- or si-(h)VDAC1-treated cells using a Nuclear/cytosol fractionation kit (Biovision, Milpitas, CA, USA) following the manufacturer’s instructions. Following centrifugation (16,000× g , 10 min), the supernatant (cytosolic fraction, Cyto), and pellet (nuclear fraction, Nuc), which was re-suspended in the original volume, were subjected to immunoblotting for KI-67 (nucleus marker), VDAC1 (cytosol fraction) and p53. Total refers to the sample collected prior centrifugation. The original WB image is in the .

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Expressing, Translocation Assay, Transfection, Cell Culture, Staining, Microscopy, Control, Western Blot, Fractionation, Centrifugation, Marker

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: Reducing VDAC1 levels inhibits p53-induced apoptosis. ( A , B ) HeLa cells were transfected with si-NT or si-(h)VDAC1 (50 nM), as described in the Methods section and 24 h post transfection, cells were transfected with empty plasmid or pCMV–p53. Approximately 48 h post transfection, VDAC1 and p53 expression levels in control and si-(h)VDAC1-treated cells were analyzed by immunoblotting ( A ) and quantified ( B ). ( C , D ) Cell death in the various samples was analyzed using annexin-V-FITC and propidium iodide (PI) staining, and FACS analysis. Representative histograms ( C ) and quantitative analysis of apoptotic cell death ( D ) are shown. Blue and black Asterix refer to significance of control and VDAC1 or control and p53, respectively. Results show means ± SEM (n = 3), * p < 0.05, *** p < 0.001. The original WB image is in the .

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Staining

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: VDAC1 inhibitor DIDS inhibits p53-induced apoptosis. HeLa cells were transfected with empty plasmid or pCMV–p53 and 24 h post transfection, cells were incubated with 70 μM DIDS. After 48 h, cell death was analyzed using PI staining, and FACS analysis. Representative histograms are presented, where the Y−axis (SSC) represents side scatter indicating cell granularity and the X-axis (FSC) represents forward scatter reflecting cell size ( A ). Quantitative analysis of apoptotic cell death ( B ). Results show means ± SEM (n = 3), ** p < 0.01.

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Transfection, Plasmid Preparation, Incubation, Staining

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: Effect of p53 on purified and mitochondria-embedded VDAC1 oligomeric state. ( A , B ) Purified VDAC1 (0.2 μM) was incubated for 5 min with reducing agent free-p53 (2 μM), obtained using the Sephadex-G-50 centrifugation-chromatography method. Then samples were incubated with the indicated concentrations of EGS for 15 min at 30 °C, followed by 10% SDS-PAGE and immunoblotting using anti-VDAC1 ( A ) or anti-p53 ( B ) antibodies. A low exposure is presented at the bottom to demonstrate the decrease in monomeric VDAC1 or p53 upon its crosslinking. ( C , D ) Purified rat mitochondria (0.5 mg/mL) were incubated for 15 min at 25 °C with the indicated concentrations of reducing agent free-p53 or without their removal (last 3 lanes), followed by EGS crosslinking and immunoblotting using anti-VDAC1 ( C ) or anti-p53 ( D ) antibodies. The final concentrations of β-mercaptoethanol (β-ME) and dithiothreitol (DTT) were 15 mM and 5 mM, respectively. The VDAC1 band labeled with the asterisk ★ points to intermolecular crosslinked VDAC1 as was previously identified as. The dimer, trimer, tetramer, and oligomer positions, as well as molecular weight standards are also indicated.

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Purification, Incubation, Centrifugation, Chromatography, SDS Page, Western Blot, Labeling, Molecular Weight

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: Overexpression of p53 in HeLa cells enhances VDAC1 expression and oligomerization. HeLa cells were transfected with empty or pCMV–p53 (2 μg) plasmid. Approximately 24 h post transfection, cells were analyzed for VDAC1 oligomerization ( A , B ) and p53 expression ( C ). Cells were harvested, washed with PBS, and incubated (3 mg/mL) with the indicated concentrations of EGS at 30 °C for 15 min and then subjected to SDS−PAGE and immunoblotting using anti−VDAC1 ( A ) or anti−p53 ( C ) antibodies. A low exposure is presented at the bottom, to demonstrate the increase in VDAC1 levels in p53-expressing cells (shown by the white arrowhead) and the decrease in monomeric VDAC1 upon its crosslinking ( A ). The positions of VDAC1 monomers to multimers and of molecular weight standards are indicated. Quantitative analysis of VDAC1 monomer and dimer levels is presented in relative units (RUs) as a function of EGS concentration ( B ).

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Incubation, SDS Page, Western Blot, Molecular Weight, Concentration Assay

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: p53 expression induces VDAC1 overexpression and oligomerization in several cell lines. HeLa cells were transfected with either empty pCMV vector or a pCMV–p53 expression plasmid (2 μg) to overexpress p53. Cells were analyzed 24 h post transfection. VDAC1 and p53 expression levels were analyzed by immunoblotting and anti−VDAC1 and anti−p53 antibodies ( A ). Quantitative analysis of VDAC1 expression levels ( B ) correspond to the mean ± SE (n = 2–6). HeLa ( C , D ), H358 ( E , F ), and A549 ( G , H ) cells were transfected with empty (2 μg) or pCMV–p53 plasmid (1 or 2 μg). 24 h post transfection, cells were analyzed for VDAC1 ( C , E , G ) and p53 ( D , F , H ) oligomerization using the indicated concentrations of EGS and immunoblotting using anti−VDAC1 or anti−p53 antibodies. A low exposure is presented at the bottom of each blot to demonstrate the decrease in monomeric VDAC1 upon its crosslinking. Quantitative analysis of VDAC1 dimers levels is presented in relative units (RUs) ( C , E , G ). The white arrows point to VDAC1- and p53-containing complexes. The positions of VDAC1 monomers to multimers and of molecular weight standards are indicated. Results show means ± SEM (n = 3), ** p < 0.01.

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Molecular Weight

Journal: Biomolecules

Article Title: p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

doi: 10.3390/biom16010141

Figure Lengend Snippet: Proposed model for p53 increases VDAC1 expression level and induces apoptosis. ( A ) Overexpression of p53 leads to activation of the VDAC1 promoter, resulting in increased VDAC1 gene expression. p53 is present and mainly mitochondrial bound, with a fraction found in the nucleus ( a ). ( B ) p53-induced mitochondria-mediated apoptosis via enhancing VDAC1 expression level and, subsequently, VDAC1 oligomerization, allowing pro-apoptotic protein release from the inter-mitochondrial space, leading to apoptotic cell death. DIDS inhibits VDAC1 oligomerization and, subsequently, p53-induced apoptosis. p53 is present and mainly mitochondrial bound, with a fraction found in the nucleus ( b ). ( C ) VDAC1 expression level influences the subcellular localization of p53. si-(h)VDAC1 decreased VDAC1 levels, reduced p53-induced VDAC1 oligomers and apoptosis, and led to nuclear accumulation of p53 ( c ), suggesting that VDAC1 regulates its mitochondrial trafficking.

Article Snippet: Logarithmically growing cells were transfected with empty or pCMV-, p53- or p53-GFP-encoding plasmids (Addgene #12091; MIT, Boston, MA, USA) using Jet-Prime transfection reagent.

Techniques: Expressing, Over Expression, Activation Assay, Gene Expression