plasmid encoding gfp  (Sino Biological)

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Volcano plot showing LC–MS/MS–based proteomic analysis of GFP pull-down samples from HEK293T cells expressing GFP-tagged wild-type (WT) or mutant LRRC8B. The x-axis represents log₂ fold change (mutant vs WT), and the y-axis shows −log₁₀ adjusted p-value. Proteins enriched in WT samples are shown on the left, while those enriched in mutant samples are shown on the right. Selected significantly enriched proteins are labeled. VDAC1 is identified as a WT-enriched interactor.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Representative immunoblot images showing VDAC protein levels in total cell lysates and isolated mitochondrial fractions from cells transfected with GFP-tagged LRRC8B WT (W) or mutant Y380S (M) constructs. Actin (∼42 kDa) is included as a loading control for total lysates. Red boxes indicate the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Isolation, Transfection, Mutagenesis, Construct, Control, Molecular Weight

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Representative immunoblot images showing protein levels of GFP-tagged LRRC8B (WT and mutant) and VDAC in input lysates used for pull-down assays. (B–C) Immunoblot analysis of four independent immunoprecipitation (IP) experiments. Bands at ∼120 kDa confirm successful pull-down of GFP-tagged LRRC8B (WT and mutant) using an anti-GFP antibody. VDAC (∼32 kDa) bands indicate co-precipitation of VDAC with LRRC8B WT and mutant proteins. VDAC levels were normalized to the corresponding LRRC8B (WT or mutant) levels for quantification. Red boxes highlight the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Molecular Weight

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Levels of the 7TGP fluorescent Wnt reporter in HEK293T cells transfected with control siRNA, Dvl1-specific siRNA, Nup358-specific siRNA, or a combination of Nup358-specific siRNA and Dvl1-specific siRNA and treated with either Wnt3a or vehicle. b. Western blot analysis and relative quantification of protein levels of Dvl1 and Axin1 in HEK293T cells transfected with either control or Nup358-specific siRNA. α-Tubulin and GAPDH were used as loading controls. Data are expressed as mean ± SD. **p ≤ 0.01. c. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with either control or Nup358-specific siRNA. d. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with control or Nup358-specific siRNA and treated with vehicle or 1,6-hexanediol 5% for 2 minutes. e. Fluorescence recovery after photobleaching (FRAP) and fusion analysis of Dvl1 condensates in HEK293T cells transfected with Nup358-specific siRNA and EGFP-Dvl1.

Article Snippet: To generate a stable Wnt reporter line, HEK293T cells were transduced with lentivirus derived from the 7TGP plasmid encoding GFP under the control of seven tandem TCF/LEF binding sites (Addgene #24305).

Techniques: Transfection, Control, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. Schematic showing the IV injection of the AAV-EGFP or AAV-sDLK1-T2A-GFP into the mice. B. Quantification of the protein levels of sDLK1 in the cortex of the mice, measured by mouse DLK1 ELISA. C. Distributions of the normalized numbers of genes up-regulated (top) and down-regulated (bottom) in different cell types of the mice injected with AAV-sDLK1-T2A-GFP, compared to mice injected with AAV-EGFP. The gene burden score is defined as the number of differentially expressed genes per 1000 UMI detected in each cell type. D. Scatter plot showing the positively correlated genes between DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected oligodendrocytes. E. Western blot of MBP (top) and MOBP (bottom). F–G. Quantification of the MBP (F) and MOBP (G) western blot. Data were analyzed by two-tailed unpaired t -test. ** p =0.0095 (MBP); *** p =0.0009 (MOBP). H. Ridge plot of the predicted chronological ages for oligodendrocytes in the mice injected with AAV-EGFP (top) and AAV-sDLK1-T2A-GFP (bottom). I. Quantification of the predicted age of oligodendrocytes. Data were reported as a box & whisker plot showing min to max and analyzed by t-test. Each dot represents a cell. **** p < 0.0001. J. UMAP plot showing the enrichment of OPC2 caused by increased sDLK1. K. Ratios of each OPC cluster. Data are reported as mean ± s.e.m. and analyzed by two-way ANOVA. * p =0.0432. L. Running enrichment score and pre-ranked list showing a negative enrichment of oligodendrocyte differentiation predicted by OPC3 markers

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: IV Injection, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, Two Tailed Test, Whisker Assay

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. UMAP plots showing eight major cell types identified in the mouse hippocampus. B. Dot plot showing expression levels of canonical cell markers in each identified cell type. C. Proportion of each cell type within animals injected with AAV-GFP and AAV-sDLK1-T2A-GFP. D. Proportion of each cell type within individual samples. E. Violin plots showing the number of unique features (left); the number of total RNA count (middle), and the percentage of mitochondrial genes (right) detected in each identified cell type. F. Correlation between UMI counts and percentage of mitochondrial genes (left) or total gene counts (right) per nuclei for each individual sample.

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. Dot plot showing the expression level of DLK1 in different cell types in the hippocampus tissue. The size of each dot represents the percentage of cells with detected DLK1 mRNA. B. Chord diagram showing DLK signaling predicted by CellChat. The lengths of the segmented outer circle reflect the expression levels of ligand proteins in each cell type and of receptor proteins in the receiving cells, showing strong expression of DLK1 signaling originating from interneurons and astrocytes to oligodendrocytes and OPCs. C. Bubble plot showing the DLK1 interactions originating from interneurons and astrocytes to different receptors. D. Venn diagram showing the overlap of upregulated DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected oligodendrocyte(top) and the overlap of downregulated DEGs in G3 Terc-/-and AAV-sDLK1-T2A-GFP injected oligodendrocyte (bottom). E. Dot plot showing the change of expression levels of myelination proteins in oligodendrocytes caused by the increase of sDLK1. F. Cnet plot showing the network of genes associated with myelination-related Gene Ontology terms and myelin-related diseases, based on enrichment analysis of the top 500 differentially expressed genes in oligodendrocytes from mice with AAV-sDLK1-T2A-GFP versus mice with AAV-GFP. Nodes represent genes or GO terms; edge colors represent the pathways each node is involved in.

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. Representative 20X images of Olig2 in the CA1 region of the hippocampus in C57BL/6 mice injected with either AAV-GFP or AAV-sDLK1-T2A-GFP. Scale bar represents 100 µm. B. Quantification of Olig2+ cell density in CA1. N = 5 mice injected with AAV-GFP and N = 4 mice injected with AAV-sDLK1-T2A-GFP. 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.9480. Data were analyzed by unpaired t-test. C. Quantification of Olig2+ cell density in CA3. N = 5 mice injected with AAV-GFP and N = 4 mice injected with AAV-sDLK1-T2A-GFP. 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.2008. Data were analyzed by unpaired t-test. D. Representative 20X images of PDGFRα in the CA1 region of the hippocampus in C57BL/6 mice injected with either AAV-GFP or AAV-sDLK1-T2A-GFP. Scale bar represents 100 µm. E. Quantification of PDGFRα+ cell density in CA1. N = 5 mice/treatment group and 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.3534. Data were analyzed by unpaired t-test. F. Quantification of PDGFRα+ cell density in CA3. N = 5 mice/treatment group and 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.6744. Data were analyzed by unpaired t-test.

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Injection

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A Venn diagram showing the overlap of upregulated (top) and downregulated (bottom) DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP -injected excitatory neurons. B. Dot plot of the top 10 Reactome pathways inferred by the upregulated overlapping DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected excitatory neurons. C–D. Running enrichment score and pre-ranked list showing a positive (C) and negative (D) enrichment of calcium ion transmembrane transport predicted by the upregulated overlapping DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected excitatory neurons. E. Schematic illustrating the experiment setup to test the chronic effects of DLK1 on neuronal activities. F. Representative fluorescence image of human iPSC-derived neurons expressing GCaMP8f showing spontaneous activity. G. Representative spontaneous calcium traces. H–J. Quantification of synchronized firing rate (H). firing amplitude (I), or spontaneous firing rate (J). K. Representative averaged calcium traces from one KCl stimulation experiment in neurons treated with DLK1 (red) and the untreated control neurons (black). Recording 400 seconds. Mean± s.e.m. L–N. Quantification of peak amplitude (L), the delayed KCl stimulation-induced neuronal responses (M), the time each neuron spent to reach peak intensity (N), from KCl stimulation-induced neuronal responses. Data are presented as mean ± s.e.m. and analyzed by unpaired t -test. ** p =0.0055 (L) **** p <0.0001 (N).

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Injection, Fluorescence, Derivative Assay, Expressing, Activity Assay, 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: 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