Journal: Cell Death & Disease

Article Title: Succinylation-dependent mitochondrial translocation of PKM2 promotes cell survival in response to nutritional stress

doi: 10.1038/s41419-018-1271-9

Figure Lengend Snippet: a VDAC3 peptide sequences were observed in the 31kDa band. Flag pull down and MS analysis of PKM2 associated proteins purified from transfected HCT116 cells. b Co-immunoprecipitation analysis showed that only VDAC3 binds directly to PKM2. c Western blot analysis of the endogenous interaction of PKM2 and VDAC3. HCT116 cell lysates were subject to immunoprecipitation with control IgG and anti-VDAC3 antibodies. d , e In vitro binding assay of purified His-tagged-PKM2 and GST-tagged-VDAC3. f K433E enhanced the interaction of PKM2 and VDAC3. Stagged-WT PKM2, a succinylation mimic K433E and a succinylation null mutant K433R and flag-VDAC3 were co-transfected into HCT116 cells for Stag bead pull down that specially pulls down Stagged proteins. g Glucose starvation enhanced the interaction of PKM2 and VDAC3. HCT116 cells were transfected with HA-tagged VDAC3, cells were harvested and subjected to Stag pull down assay after 8 h glucose starvation. h Western blot analysis of VDAC3 and PKM2 in HCT116 cell lysates after RNA-interference-mediated knockdown of PKM2. i Ectopic expression of Flag-PKM2 in HCT116 cells upregulates VDAC3 in a dose-dependent manner. Treatment with the proteasome inhibitor MG132 at 10 μM for 8 h also caused VDAC3 upregulation. j Expression levels of VDAC3 and PKM2 protein analyzed with immunoblotting in PKM2 control (shControl) and PKM2 knockdown (shPKM2). Cells were treated with cycloheximide (CHX, 150 µg/ml) at indicated times. k In vivo VDAC3 ubiquitination assay. Cellular level VDAC3 ubiquitination assays were carried out in PKM2 WT or knockout MEFs. Cells were co-transfected with His-HA-ubiquitin (Ub) and GFP-Parkin, immunoprecipitated with VDAC3 antibody and immunoblotted with antibodies to HA, VDAC3, PKM2, or GFP. Cells were treated with MG132 (10 μM) for 8 h before harvest. l In vivo VDAC ubiquitination assay. Cellular level VDAC ubiquitination assays were carried out in HCT116 cells. Cells were co-transfected with His-HA-ubiquitin (Ub), Flag-VDAC3, GFP-Parkin, and cMyc-PKM2 plasmids, immunoprecipitated with Flag beads and immunoblotted with antibodies to HA and Flag. Cells were treated with MG132 (10 μM) for 8 h before harvest

Article Snippet: PKM2 (D78A4) XP ® Rabbit mAb (#4053, CST) and VDAC3 Rabbit pAb (bs-7647R, Bioss) were used to blot PKM2 and VDAC3 separately.

Techniques: Purification, Transfection, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Mutagenesis, Pull Down Assay, Expressing, In Vivo, Ubiquitin Assay, Knock-Out

Journal: Cell Death & Disease

Article Title: Succinylation-dependent mitochondrial translocation of PKM2 promotes cell survival in response to nutritional stress

doi: 10.1038/s41419-018-1271-9

Figure Lengend Snippet: a Outer mitochondrial membrane permeability as determined by the NADH oxidation rate in isolated mitochondria from HCT116 cells after 0 or 25 mM glucose treatment for 6 h. b RNA-interference-mediated knockdown of PKM2 or VDAC3 caused a decrease in the rate of NADH oxidation. Mitochondria isolated from HCT116 cells were used for NADH oxidation assays. c Mitochondria from PKM2 and VDAC3 knockdown HCT116 cells were analyzed for COX activity after 12 h glucose starvation. Data are presented as mean ± SEM of three independent experiments. d PKM2 and VDAC3 maintain cellular ATP levels. ATP levels of indicated HCT116 cells were measured using the Cell Titer-Glo Luminescent Cell Viability Assay. e K433E enhances the NADH oxidation rate. Stagged-PKM2 and the succinylation mimic K433E were co-transfected into HCT116 cells for NADH oxidation rate assays

Article Snippet: PKM2 (D78A4) XP ® Rabbit mAb (#4053, CST) and VDAC3 Rabbit pAb (bs-7647R, Bioss) were used to blot PKM2 and VDAC3 separately.

Techniques: Permeability, Isolation, Activity Assay, Cell Viability Assay, Transfection

Journal: Cell Death & Disease

Article Title: Succinylation-dependent mitochondrial translocation of PKM2 promotes cell survival in response to nutritional stress

doi: 10.1038/s41419-018-1271-9

Figure Lengend Snippet: a PKM2 and VDAC3 promote cell survival under nutritional stress. Survival of HCT116 cells in glucose-free media was measured with the Trypan blue exclusion method. b , c VDAC3 promotes tumor development. Tumor volume of VDAC3-kd HCT116 xenografts ( n = 5). d PKM2 and VDAC3 are upregulated in human colon cancer. Fresh colon cancer and matched surrounding normal tissue from the same given patient were homogenized, and cytosolic and mitochondria fractions were separated. PKM2 was determined by western analysis. N (normal tissue), T (colon cancer). e Positive correlation of mitochondrial PKM2 and VDAC3 protein levels in human colon cancer. Statistical significance was determined with the χ 2 -test. R is the correlation coefficient. f , g Mitochondrial PKM2 and VDAC3 increased before solid tumor formation. f Sketch outlining the AOM/DSS-induced CRC mode. C57 mice were treated 1× with or without AOM, followed by periodic administration of 2% DSS in water. n = 12 per group. g Intestine issues were analyzed for mitochondrial PKM2 and VDAC3

Article Snippet: PKM2 (D78A4) XP ® Rabbit mAb (#4053, CST) and VDAC3 Rabbit pAb (bs-7647R, Bioss) were used to blot PKM2 and VDAC3 separately.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Succinylation-dependent mitochondrial translocation of PKM2 promotes cell survival in response to nutritional stress

doi: 10.1038/s41419-018-1271-9

Figure Lengend Snippet: a Compound 8 blocks glucose starvation induced PKM2 mitochondrial translocation. Isolation of mitochondria and immunoblot of PKM2, tubulin and Tom 40 were carried out with or without compound 8 treatment. b Compound 8 disrupts interaction of PKM2 and VDAC3. HCT116 cells were transfected with HA-tagged PKM2, cells were harvested and subjected to stag pull down after 8 h 20 µM compound 8 treatment. c Compound 8 induces VDAC3 for degradation. cells were harvested and evaluated VDAC3 protein levels after 8 h 10 or 20 µM compound 8 treatment. d , e Compound 8 caused a decrease in the rate of NADH oxidation. After 8 h of treatment with 20 µM compound 8, HCT116 cells mitochondria were isolated for NADH oxidation assays. NAD/NADH ratios in HCT116 cell lysates were evaluated by LC–MS. f Compound 8 inhibited ATP production in HCT116 cells. g The tumor volume ( n = 7) of HCT116 xenografts from mice with intraperitoneal injection of compound 8 and vehicle control

Article Snippet: PKM2 (D78A4) XP ® Rabbit mAb (#4053, CST) and VDAC3 Rabbit pAb (bs-7647R, Bioss) were used to blot PKM2 and VDAC3 separately.

Techniques: Translocation Assay, Isolation, Western Blot, Transfection, Liquid Chromatography with Mass Spectroscopy, Injection

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: KEGG pathway clustering analysis of different comparison groups, the PPI network of differentially lactylated proteins in the pathways of interest, and verification of Vdac3 protein lactylation modifications. (A) Heatmap of lactylation sites for 12 key proteins. Each row represents a differentially modification site; each column represents a sample. Red indicates a high-level modification, blue indicates a low-level modification, and grey indicates sites that cannot be quantified in the corresponding samples. (B) The horizontal axis depicts different comparison groups, whereas the vertical axis indicates the enriched KEGG pathways. The colored blocks represent the functional descriptions of differentially expressed modified proteins enriched in each comparison group. Blue signifies high enrichment significance, whereas blue-white signifies low enrichment significance. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) PPI network of differentially lactylated proteins involved in the 4-VO group and sham group in the five pathways of interest. (D) PPI network in the 4-VO group and 4-VO + EA group. The color of the outer circle indicates the corresponding KEGG pathway of the protein, whereas the shape within the circle denotes the upregulation or downregulation of the modification sites contained within the protein. A diamond shape signifies proteins with upregulated modification sites, a downward arrow indicates proteins with downregulated modification sites, and a circle signifies proteins with upregulated and downregulated modification sites. The red box indicates the Vdac3 protein. (E) The IP experiment verified the lactylation modification level of Vdac3. (F) Quantification was performed using ImageJ software, and the values are expressed as the mean with the corresponding standard error from three replicate experiments ( n = 3, compared with the sham group, ## p < 0.01; compared with the 4-VO group, ** p < 0.01). 4-VO, four-vessel occlusion; EA, electroacupuncture; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction; IP, immunoprecipitation; Vdac3, mitochondrial voltage-dependent anion channel protein3.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Comparison, Modification, Functional Assay, Software, Immunoprecipitation

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: Analysis of lactylation site motifs in different groups and representative mass spectra of key proteins. (A) A heatmap of amino acids adjacent to lysine lactylation sites, with a green/red scale (−5 to 5) indicating the frequency of amino acid detection; red indicates high frequency; green indicates low frequency. The height ratio of amino acid abbreviation letters at specific positions reflects motif characteristics, with a % difference greater than 0 indicating a higher frequency of that amino acid at that position compared to the background, and less than 0 indicating a lower frequency. (B) Four conserved motif sites of lysine. (C) Sequence motif map of lactylation modification, 10 amino acids upstream and downstream of the modification site were selected, and the vertical axis indicates the difference between the proportion of the various amino acids at the same site in the experimental data set and the reference background, that is, percentage difference (%difference). The height ratio of amino acid abbreviation letters at specific positions represents the motif characteristics, and a %difference greater than 0 indicates that the frequency of the amino acid at this position is higher than the background. Values lower than 0 indicate that the frequency of the amino acid at this position is lower than the background. (D–F) The lactylation site of Vdac3 was identified by mass spectrometry. PPI, protein–protein interaction; 4-VO, four-vessel occlusion; EA, electroacupuncture.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Sequencing, Modification, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells

doi: 10.1074/jbc.M112.433847

Figure Lengend Snippet: VDAC knockdown decreases mitochondrial membrane potential in HepG2 cells. In A, relative abundance of mRNA for VDAC1, VDAC2 and VDAC3 in native HepG2 cells was determined by qPCR. In B, cells were transfected with non-target siRNA and siRNA against VDAC1, VDAC2 and VDAC3. After 48 h, mRNA expression of the corresponding VDAC isoform and protein expression of all three isoforms were assessed by qPCR and immunoblotting. In C at 48 h after transfection with non-target siRNA and siRNA against VDAC1, VDAC2, and VDAC3, HepG2 cells were loaded with TMRM and imaged. Note decrease of mitochondrial TMRM fluorescence after knockdown of each VDAC isoform, but most markedly after VDAC3 knockdown. Image intensity was pseudocolored according to the reference bar. Arrows identify 4-μm fiduciary fluorescent beads. In D, average TMRM fluorescence after knockdown was plotted in comparison to transfection with non-target siRNA. *, p < 0.05 from five independent experiments analyzing 4–5 random fields containing 5–10 cells.

Article Snippet: Blots were blocked for 60 min in 5% albumin and probed with antibodies against VDAC1 (SC-8828, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); VDAC2 (Ab-47104, Abcam, Cambridge, MA, 1:500); VDAC3 (MSA03/E0836, MitoSciences, Eugene, OR, 1:1000); and β-actin (691002, MP Biomedicals, Solon, OH, 1:2000).

Techniques: Knockdown, Membrane, Transfection, Expressing, Western Blot, Fluorescence, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells

doi: 10.1074/jbc.M112.433847

Figure Lengend Snippet: VDAC3 knockdown decreases ATP, phosphorylation potential, adenine nucleotides, and NAD(P)H redox state. In A--C, adenine nucleotides and phosphate in HepG2 cells were measured after siRNA knockdown of VDAC1, VDAC2, and VDAC3, as described under “Experimental Procedures.” In D, NAD(P)H autofluorescence was imaged by multiphoton microcopy. Note that VDAC3 but not VDAC1 or VDAC2 knockdown decreased mitochondrial NADH autofluorescence. In E, average NAD(P)H autofluorescence after knockdown of VDAC isoforms is plotted in comparison to transfection with non-target siRNA. In F, NAD(P)H autofluorescence before and after addition of CCCP plus oligomycin or myxothiazol was measured for HepG2 cells transfected with non-target siRNA and siRNA against VDAC3. *, p < 0.05 compared with Non-target or Baseline from three independent experiments analyzing 4–5 random fields containing 5–10 cells.

Article Snippet: Blots were blocked for 60 min in 5% albumin and probed with antibodies against VDAC1 (SC-8828, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); VDAC2 (Ab-47104, Abcam, Cambridge, MA, 1:500); VDAC3 (MSA03/E0836, MitoSciences, Eugene, OR, 1:1000); and β-actin (691002, MP Biomedicals, Solon, OH, 1:2000).

Techniques: Knockdown, Phospho-proteomics, Comparison, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells

doi: 10.1074/jbc.M112.433847

Figure Lengend Snippet: VDAC 1, 2, and 3 isoforms have different sensitivity to blockade by tubulin. Double knockdowns of VDAC isoforms were performed in all possible combinations in HepG2 cells, and the remaining VDAC isoform was isolated and inserted into lipid bilayers. In A, normalized average conductance, G/Gmax, is plotted versus applied voltage for the three VDAC isoforms in the absence of tubulin. In B, tubulin (10 nm) was added to both sides of the membrane, which increased VDAC voltage-induced closure and decreased minimum conductance at > ± 40 mV for VDAC1 and 2 but did not affect VDAC3. In C, normalized conductance at −50 mV was plotted for all VDAC isoforms at tubulin concentrations of 10 nm and 50 nm, as indicated. *, p < 0.001 compared with no tubulin from 3–6 experiments for each group.

Article Snippet: Blots were blocked for 60 min in 5% albumin and probed with antibodies against VDAC1 (SC-8828, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); VDAC2 (Ab-47104, Abcam, Cambridge, MA, 1:500); VDAC3 (MSA03/E0836, MitoSciences, Eugene, OR, 1:1000); and β-actin (691002, MP Biomedicals, Solon, OH, 1:2000).

Techniques: Isolation, Membrane