Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: Identification of the protein target of ML405/85 (A) Venn diagram of protein peptide profiles identified by DARTS in DMSO-, ML405-, and 1685-treated cells; 1,016 proteins had essentially the same peptide profile in all three samples, whereas 69 proteins had a different profile from the DMSO control sample. (B) Representative images for the lack of an effect of candidate target protein expression (green) on the cholesterol storage (red) of NPC3 patient cells. (C) Representative images showing the effects of transient expression (left panel) or suppression (right panel) of HSP candidates ERP29, HYOU1, and TRAP1 on cholesterol storage in NPC3 patient cells or mouse NPC1 cells. Left panel: cells expressing candidate proteins are red and cholesterol is green. Right panel: cells in which protein is silenced are green, cholesterol is red. (D) Representative images showing expression of TRAP1 (green) in Fabry disease patient cells decreases the Gb3 storage as determined by VTB staining (red). Likewise, the expression of TRAP1 (red) in Farber and Wolman disease cells decreases the cholesterol storage (green; OlyA) in these cells. Images are representative of at least three independent experiments. Graphs indicate the percentage of transfected cells corrected for lipid storage ( n = 50–75) and are represented as mean ± SD Scale bar, 50 μm.

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Control, Expressing, Staining, Transfection

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: List of potential protein targets for the ML405/1685 compounds

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Membrane, Ubiquitin Proteomics

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: ML405/85 mimic TRAP1 actions on mitochondrial respiratory chain enzymes and metabolites (A) Treatment with ML405 inhibits phosphorylation of transiently expressed mitochondria-targeted c-Src at Tyr-416 (p-Y416) but has no effect on total mitochondrial c-Src (Input) in ARPE cells. The blot shown is representative of four independent experiments. (B) Densitometry of phospho- c -Src bands in (A) normalized to total c-Src (Input). (C and D) ML405/85 inhibit complex II (SDH) activity of the mitochondrial respiratory chain in purified ARPE mitochondria (C), which results in the expected increase in cellular succinate levels (D). TTFA: thenoyltrifluoroacetone, complex II inhibitor (positive control). (E) Treatment with ML405 lowers COX activity in purified ARPE mitochondria. (F) Treatment with ML405/85 increase cellular and secreted lactate levels in ARPE cells. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001.

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Phospho-proteomics, Activity Assay, Purification, Positive Control

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: TRAP1 agonists ameliorate mitochondrial and ER stress (A and B) ML405 decreases the red/green fluorescence ratio in Mitotimer-expressing NPC1 cells, indicating reduced mitochondrial oxidative stress (A, representative image of cell mitochondria quantified in B). (C) Treatment with ML405/85 reduce superoxide levels (red; MitoSox) in isogenic wt and NPC1-null cells but have no effect in isogenic TRAP1-null cells, indicating that the reduction of mitochondrial superoxide is mediated by TRAP1. (D) Treatment with ML405 increases ATP levels in both NPC3 and Fabry patient cells. (E) Treatment with ML405 increases the residual α-galactosidase activity in Fabry patient cells to varying degrees based on the specific α-galactosidase mutation of each cell line. (F–H) Similarly, treatment with ML405 increases the activity of lysosomal acid lipase in Wolman (F), tripeptidyl peptidase 1 in CLNII (G), and galactocerebrosidase in Krabbe (H) patient cells. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001.

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Fluorescence, Expressing, Activity Assay, Mutagenesis

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: Mitochondrial dysfunction affects lysosomal function (A) Representative images showing that cell treatment with mitochondrial respiratory chain inhibitors antimycin A (complex III) or atpenin A5 (SDH) induces the lysosomal lipid storage in wt cells and exacerbates the storage in NPC1 cells. (B) Quantitation of the lipid storage seen in wt and NPC1 cells (A). At least 150 cells were quantitated for each sample and each experiment was repeated three times. Images were taken using the same exposure settings. (C) Schematic showing the importance of mTORC1 as a sensor of glucose availability and a master regulator of mitochondria and lysosome/autophagy function. (D) Treatment with 1685 or exogenous TRAP1 expression increases the levels of cellular AMPK and phosphorylated AMPK (pAMPK). GAPDH: glyceraldehyde 3-phosphate dehydrogenase. (E) Effect of ML405/85 on pAMPK levels is rapid, reaching a maximum at about 1 h following treatment. Metform: metformin, known inducer of pAMPK (positive control). (F) Densitometry of pAMPK bands from (E) normalized to GAPDH signal. (G) Treatment with 1685 inhibits phosphorylation of the mTOR target p70S6 kinase in a rapid manner. Graph represents levels of normalized phosphorylated p70S6 kinase relative to total p70S6 kinase protein levels. The blots shown are representative of three independent experiments. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001, ∗ p < 0.05.

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Quantitation Assay, Expressing, Positive Control, Phospho-proteomics

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: TRAP1 agonists show efficacy in a mouse Fabry model Age-matched Fabry mice treated with 30 mg/kg ML405 three times/week for four weeks showed a reduced Gb3 storage in the liver (A) plasma (B) kidney (C), and heart (D). Values shown are the average of three replicates, n = 9–10. ∗∗∗ p < 0.0005, ∗∗ p < 0.001, ∗ p < 0.05.

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Clinical Proteomics

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet:

Article Snippet: TRAP1 Human shRNA kit , Origene , Cat#:TG300868.

Techniques: Virus, Recombinant, Cytochrome c Oxidase Assay, Viability Assay, Protein Extraction, BIA-KA, Isolation, Mutagenesis, Variant Assay, Knock-Out, CRISPR, Sequencing, shRNA, Plasmid Preparation, Software, Microscopy

Journal: bioRxiv

Article Title: The Cancer/Testis Antigen FATE1 Antagonizes Fission and Preserves Mitochondrial Network Integrity under Cytotoxic Stress

doi: 10.1101/2025.08.18.670752

Figure Lengend Snippet: a Sequence alignment of the C-terminal regions of human FATE1 and Mff generated with Jalview using the T-Coffee algorithm under default settings. Amino acid numbering corresponds to Mff. b Coiled coil domain prediction of FATE1 using the COILS program. Mff and Bnip3 were included as positive and negative controls, respectively. Predicted probabilities for coiled coil formation are shown for scanning windows of 14, 21 and 28 residues. c Schematic comparison of the domain architecture of Mff and FATE1, highlighting sequence homologies, including the predicted coiled coil domain and transmembrane region. R1 and R2 indicate the short repeat motifs in Mff involved in Drp1 recruitment. d Representative widefield images of GFP-Ctrl, GFP-FATE1, or GFP-Mff co-expressed with RFP-KDEL (ER marker) for 24 h in HeLa cells. Blue arrows, ER-localized FATE1 at perinuclear regions. e Representative widefield images of GFP-Ctrl, GFP-FATE1, or GFP-Mff co-expressed with mito-RFP (mitochondrial marker) for 24 h in HeLa cells. f Subcellular fractionation of HeLa cells transfected with GFP-tagged constructs as indicated. Fractionation yielded cytosolic (S1), rest-mitochondrial (P1) and purified mitochondrial (P2) fractions, analyzed by immunoblotting. g Representative widefield images of MCF-7 cells expressing GFP-FATE1 or GFP-Mff for 24 h and IF stained for mitochondrial matrix protein TRAP1. Plot profiles along yellow lines in zoom images show fluorescence intensity of GFP and TRAP1 signals. h STED super-resolution microscopy of HeLa cells expressing pcDNA-FATE1 and IF stained for FATE1 in combination with Cox IV (IMM marker) or Tom20 (OMM marker). Plot profiles show fluorescence intensity of FATE1 and Cox IV or Tom20. Scale bars, 10 µm

Article Snippet: For immunofluorescence, cells were then incubated with primary antibodies against Tom20 (Santa Cruz Biotechnology, no. sc-11415 or sc-17764; 1:200) for 1 h at room temperature, or against COX IV (Cell Signaling, no. 4850S; 1:250), or TRAP1 (Novus Biologicals, no. NB300-555; 1:100) antibodies at 4°C overnight.

Techniques: Sequencing, Generated, Comparison, Marker, Fractionation, Transfection, Construct, Purification, Western Blot, Expressing, Staining, Fluorescence, Super-Resolution Microscopy

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: Identification of the protein target of ML405/85 (A) Venn diagram of protein peptide profiles identified by DARTS in DMSO-, ML405-, and 1685-treated cells; 1,016 proteins had essentially the same peptide profile in all three samples, whereas 69 proteins had a different profile from the DMSO control sample. (B) Representative images for the lack of an effect of candidate target protein expression (green) on the cholesterol storage (red) of NPC3 patient cells. (C) Representative images showing the effects of transient expression (left panel) or suppression (right panel) of HSP candidates ERP29, HYOU1, and TRAP1 on cholesterol storage in NPC3 patient cells or mouse NPC1 cells. Left panel: cells expressing candidate proteins are red and cholesterol is green. Right panel: cells in which protein is silenced are green, cholesterol is red. (D) Representative images showing expression of TRAP1 (green) in Fabry disease patient cells decreases the Gb3 storage as determined by VTB staining (red). Likewise, the expression of TRAP1 (red) in Farber and Wolman disease cells decreases the cholesterol storage (green; OlyA) in these cells. Images are representative of at least three independent experiments. Graphs indicate the percentage of transfected cells corrected for lipid storage ( n = 50–75) and are represented as mean ± SD Scale bar, 50 μm.

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Control, Expressing, Staining, Transfection

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: List of potential protein targets for the ML405/1685 compounds

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Membrane, Ubiquitin Proteomics

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: ML405/85 mimic TRAP1 actions on mitochondrial respiratory chain enzymes and metabolites (A) Treatment with ML405 inhibits phosphorylation of transiently expressed mitochondria-targeted c-Src at Tyr-416 (p-Y416) but has no effect on total mitochondrial c-Src (Input) in ARPE cells. The blot shown is representative of four independent experiments. (B) Densitometry of phospho- c -Src bands in (A) normalized to total c-Src (Input). (C and D) ML405/85 inhibit complex II (SDH) activity of the mitochondrial respiratory chain in purified ARPE mitochondria (C), which results in the expected increase in cellular succinate levels (D). TTFA: thenoyltrifluoroacetone, complex II inhibitor (positive control). (E) Treatment with ML405 lowers COX activity in purified ARPE mitochondria. (F) Treatment with ML405/85 increase cellular and secreted lactate levels in ARPE cells. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001.

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Phospho-proteomics, Activity Assay, Purification, Positive Control

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: TRAP1 agonists ameliorate mitochondrial and ER stress (A and B) ML405 decreases the red/green fluorescence ratio in Mitotimer-expressing NPC1 cells, indicating reduced mitochondrial oxidative stress (A, representative image of cell mitochondria quantified in B). (C) Treatment with ML405/85 reduce superoxide levels (red; MitoSox) in isogenic wt and NPC1-null cells but have no effect in isogenic TRAP1-null cells, indicating that the reduction of mitochondrial superoxide is mediated by TRAP1. (D) Treatment with ML405 increases ATP levels in both NPC3 and Fabry patient cells. (E) Treatment with ML405 increases the residual α-galactosidase activity in Fabry patient cells to varying degrees based on the specific α-galactosidase mutation of each cell line. (F–H) Similarly, treatment with ML405 increases the activity of lysosomal acid lipase in Wolman (F), tripeptidyl peptidase 1 in CLNII (G), and galactocerebrosidase in Krabbe (H) patient cells. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001.

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Fluorescence, Expressing, Activity Assay, Mutagenesis

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: Mitochondrial dysfunction affects lysosomal function (A) Representative images showing that cell treatment with mitochondrial respiratory chain inhibitors antimycin A (complex III) or atpenin A5 (SDH) induces the lysosomal lipid storage in wt cells and exacerbates the storage in NPC1 cells. (B) Quantitation of the lipid storage seen in wt and NPC1 cells (A). At least 150 cells were quantitated for each sample and each experiment was repeated three times. Images were taken using the same exposure settings. (C) Schematic showing the importance of mTORC1 as a sensor of glucose availability and a master regulator of mitochondria and lysosome/autophagy function. (D) Treatment with 1685 or exogenous TRAP1 expression increases the levels of cellular AMPK and phosphorylated AMPK (pAMPK). GAPDH: glyceraldehyde 3-phosphate dehydrogenase. (E) Effect of ML405/85 on pAMPK levels is rapid, reaching a maximum at about 1 h following treatment. Metform: metformin, known inducer of pAMPK (positive control). (F) Densitometry of pAMPK bands from (E) normalized to GAPDH signal. (G) Treatment with 1685 inhibits phosphorylation of the mTOR target p70S6 kinase in a rapid manner. Graph represents levels of normalized phosphorylated p70S6 kinase relative to total p70S6 kinase protein levels. The blots shown are representative of three independent experiments. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001, ∗ p < 0.05.

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Quantitation Assay, Expressing, Positive Control, Phospho-proteomics

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: TRAP1 agonists show efficacy in a mouse Fabry model Age-matched Fabry mice treated with 30 mg/kg ML405 three times/week for four weeks showed a reduced Gb3 storage in the liver (A) plasma (B) kidney (C), and heart (D). Values shown are the average of three replicates, n = 9–10. ∗∗∗ p < 0.0005, ∗∗ p < 0.001, ∗ p < 0.05.

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Clinical Proteomics

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet:

Article Snippet: TRAP1 (Myc-DDK-tagged)-Human TNF receptor-associated protein 1 , Origene , Cat#RC203439.

Techniques: Virus, Recombinant, Cytochrome c Oxidase Assay, Viability Assay, Protein Extraction, BIA-KA, Isolation, Mutagenesis, Variant Assay, Knock-Out, CRISPR, Sequencing, shRNA, Plasmid Preparation, Software, Microscopy

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A)TRAP1 expression levels in normal versus colon tumor tissues, according to the TCGA database, as displayed on the GEP2 website. (B) TRAP1 protein expression was not detectable in TRAP1 CRISPR/Cas9 CT26 cells using Western blot analysis. (C) TRAP1 mRNA levels were quantified by qPCR in WT and KO colon cancer cells. (D) Cell growth of WT and KO cells was measured at 24 h, 48 h, 72 h using CCK-8. (E) GSEA of proteomic data using Molecular Signatures Database (MSigDB) GO BP gene set is summarized as the normalized enrichment score (NES) in WT and KO cells. (F) GSEA plots of cytoplasm translation, regulation of cell cycle G1-S, detoxification and cellular oxidant detoxification gene signatures that are associated with depletion of TRAP1. (G) Intracellular ROS levels in WT and KO cells were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. (H) Quantitation of ROS. (I) Cell lysates were prepared from WT EV, KO2 EV and KO2 TOE for Western blot analysis to detect TRAP1 and Actin proteins. (J) Intracellular ROS levels in WT EV, KO2 EV and KO2 TOE were detected using an ROS assay kit and analyzed by flow cytometry. (K) Quantitation of ROS. P < 0.05 (*), P < 0.01 (**). (L) WT and individual KO cells were collected and stained with PI. The cell cycle was analyzed using flow cytometry and BD software. (M) Cells were seeded in 6-well plates and cultured for 7 days. Cells were stained with crystal violet, and colony numbers were quantified in (N).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: Expressing, CRISPR, Western Blot, CCK-8 Assay, ROS Assay, Flow Cytometry, Quantitation Assay, Staining, Software, Cell Culture

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A, B) The pH value of the culture medium was measured in WT and KO CT26 cells. (C, D) Enrichment analysis of proteomic data was performed using the GSEA MSigDB GO BP gene set. GSEA plots and heat maps for pH regulation genes in the TRAP1 metabolic gene signature. (E) GSEA of mass spectrometry was performed with MSigDB GO CC gene set and is summarized as the normalized enrichment score (NES) in WT and KO cells. (F) GSEA plots for NADH dehydrogenase complex and cytosolic ribosome are presented within the TRAP1 metabolic gene signature. (G, H) The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of WT and KO cells were measured using the Seahorse assay. The data were analyzed using Wave software. (I) The energy map generated from the Seahorse assay in WT and KO cells is shown. The empty and dotted squares represent duplicate experiments within each group. Blue indicates the WT group, red represents the KO6 group, and green represents the KO7 group. (J, K) Glucose consumption and lactate production were measured in WT and KO cells using Glucose-GLO and Lactate-GLO kits, respectively. (L) Illustration of TRAP1 depletion favors the glycolysis pathway than oxidative phosphorylation in colon cancer. P < 0.05 (*), P < 0.01 (**).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: Mass Spectrometry, Software, Generated, Phospho-proteomics

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A) TRAP1, PDK1, pPDH (S232), pPDH (S293), PDH, and Actin protein levels were detected in WT and individual KO cells using Western blot analysis. (B) WT and individual KO cells were fixed with 4 % paraformaldehyde and stained with pPDH (S232) antibody. Staining was analyzed using a Nikon microscope. (C) WT and KO cells were treated with various concentrations of rotenone for 48 h. Cell viability was determined using the CCK-8 assay at OD450. (D) Cells were treated with either Ctrl or 200 nM rotenone for 24 h. Intracellular ROS levels were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. Quantitation of intracellular ROS levels is shown in (E). (F, G) Glucose consumption and lactate production were measured in cells treated with 10 nM or 50 nM rotenone using Glucose-GLO and Lactate-GLO kits, respectively. (H) WT and KO cells were treated with either Ctrl, 1.25 mM, or 2.5 mM 2DG for 7 days. Cells were stained with crystal violet, and colony numbers were quantified in (I). (J) WT and KO cells were treated with various concentrations of 2DG for 48 h. Cell viability was determined using the CCK-8 assay at OD450. (K) WT and KO cells were treated with Ctrl or 5 mM 2DG for 24 h. Cellular ROS levels were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. Quantitation of cellular ROS levels is shown in (L). (M) WT and KO cells were treated with 0.625 mM, 1.25 mM, or 2.5 mM 2DG for 24 h. The culture medium was collected, and glucose consumption was analyzed using Promega Glucose-GLO kits. (N) WT and KO cells were treated with various concentrations of 2DG for 48 h. Cell lysates were prepared for Western blot analysis to detect TRAP1, CHOP, PARP, and Actin protein levels. P < 0.05 (*), P < 0.01 (**).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: Western Blot, Staining, Microscopy, CCK-8 Assay, ROS Assay, Flow Cytometry, Quantitation Assay

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A) GSEA of mass spectrometry was performed with MSigDB HALLMARK gene set and is summarized as the normalized enrichment score (NES) in WT and KO cells. (B, C) Enrichment analysis was carried out using the GSEA HALLMARK gene set. GSEA plots of hypoxia and glycolysis genes are presented within the TRAP1 metabolic gene signature. (D) Differential gene expressions were analyzed using DEseq2, with fold change >1.5 and FDR <0.1. Volcano plot shows relative fold change (log2) in protein abundance versus −log10(P values) from WT cells compared with KO cells. Proteins that demonstrate a significant change in expression are colored, with decreased expression in green color and increased expression in red color. (E, F) BP and KEGG pathway analysis of the differential gene expressions was conducted using ShinyGO website. The dot plot represents the top 10 significant pathways ranked according to − log enrichment P value. (G) Correlation analysis of TRAP1 with HIF1A and HIF1A with MCT1 expression in the TCGA database was performed using the ENCORI website. (H) Cells were fixed with 4 % paraformaldehyde and stained with HIF1A antibody. Staining was analyzed ushing a Nikon microscope. (I) Nuclear and cytoplasmic fractions of WT and KO cells were separated using a nuclear extraction kit (ThermoFisher). TRAP1, HIF1A, Lamin B1, and Tubulin proteins were detected using specific antibodies by Western blotting. (J) WT and KO cells stably expressing HRE-luciferase were subjected to a reporter assay using the Promega luciferase kit, One-GLO. Luminescence levels were measured using a Luminescence reader. (K) TRAP1, HIF1A, GLUT1, MCT1, and Actin proteins were detected using Western blotting. (L) Illustration of TRAP1 depletion induces ROS generation to facilitate glycolysis pathway through ROS-HIF1A axis. P < 0.05 (*), P < 0.01 (**).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: Mass Spectrometry, Quantitative Proteomics, Expressing, Staining, Microscopy, Extraction, Western Blot, Stable Transfection, Luciferase, Reporter Assay

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A) Cell lysates were prepared from WT EV, KO2 EV, KO2 TOE, KO6 EV, and KO6 TOE cells. TRAP1, PDK1, pPDH (S232), pPDH (S293), PDH, and Actin proteins were detected using Western blot analysis. (B) Cells were fixed with 4 % paraformaldehyde and stained with pPDH (S232) antibody. Staining was analyzed using a Nikon microscope. (C) Cells were treated with different dosages of rotenone for 48 h. Cell viability was analyzed using the CCK-8 assay at OD450. (D) WT EV, KO2 EV and KO2 TOE cells were treated with Ctrl and 200 nM rotenone for 24 h. Intracellular ROS levels of were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. (E) Quantitation of intracellular ROS levels. (F) Cells were fixed with 4 % paraformaldehyde and stained with HIF1A antibody. Staining was analyzed using a Nikon microscope. (G) Cell lysates were prepared from WT EV, KO2 EV and KO2 TOE cells. TRAP1, GLUT1, MCT1, and Actin protein levels were detected using western blot analysis. (H, I) The relative glucose consumption rate and lactate concentration of the culture medium at different time points in WT EV, KO2 EV and KO2 TOE cells were measured using Glucose-GLO and Lactate-GLO kits. (J) Cells were treated with 5 mM 2DG for 24 h. Intracellular ROS levels were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. (K) Quantitation of intracellular ROS levels. (L) Cells were treated with different dosages of 2DG for 48 h. Cell viability was analyzed using the CCK-8 assay at OD450. P < 0.05 (*), P < 0.01 (**).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: Western Blot, Staining, Microscopy, CCK-8 Assay, ROS Assay, Flow Cytometry, Quantitation Assay, Concentration Assay

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A) WT and KO cells were treated with 2 mM Trolox for 2 h. Intracellular ROS levels were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. (B) Quantitation of intracellular ROS levels were performed. (C) Cells were treated with Ctrl and 1 mM Trolox for 24h. Cells were fixed with 4 % paraformaldehyde and stained with HIF1A antibody. Staining was analyzed using a Nikon microscope. (D) WT and KO cells stably expressing HRE-luciferase were treated with various concentrations of Trolox for 18 h. Reporter assays were performed using One-GLO. (E) Cells were treated with 0.3 mM or 1 mM Trolox for 24 h. Cell lysates were prepared for Western blot analysis to detect TRAP1, HIF1A, GLUT1, MCT1, and Actin proteins. (F) Cells were treated with 0.3 mM or 1 mM Trolox for 48 h. The pH value of the culture medium was measured using a pH meter. (G, H) Cells were treated with Ctrl, 2 mM Trolox for 24 h. Glucose consumption and lactate production in Trolox-treated cells were measured using Glucose-GLO and Lactate-GLO kits. (I) Cells were treated with Ctrl, 50 μM, or 100 μM PX478 for 24 h. Cell lysates were prepared for Western blot analysis to detect TRAP1, HIF1A, GLUT1, MCT1, and Actin proteins. (J, K) Glucose consumption and lactate production in 100 μM PX-478-treated cells were measured using Glucose-GLO and Lactate-GLO kits. (L) Cells were treated with 100 μM PX478 for 24 h. Cells were fixed with 4 % paraformaldehyde and stained with pPDH(S232) antibody. Staining was analyzed using a Nikon microscope. (M) Cells were treated with 100 μM PX478 for 48 h. Cell lysates were prepared for Western blot analysis to detect TRAP1, HIF1A, PDK1, pPDH(S232), PDH, and Actin proteins. P < 0.05 (*), P < 0.01 (**).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: ROS Assay, Flow Cytometry, Quantitation Assay, Staining, Microscopy, Stable Transfection, Expressing, Luciferase, Western Blot

Journal: Cancer letters

Article Title: Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer

doi: 10.1016/j.canlet.2025.217977

Figure Lengend Snippet: (A) Cells were treated with 20 mM DCA for 48 h. TRAP1, PDK1, pPDH (S232), pPDH (S293), PDH, and Actin protein levels were detected using Western blot analysis. (B) Cells were treated with Ctrl and 20 mM DCA for 24 h. Cells were fixed with 4 % paraformaldehyde and stained with pPDH (S232) antibody. Staining was analyzed using a Nikon microscope. (C, D) The pH value of the culture medium was measured in cells treated with various concentrations of DCA. (E, F) The relative glucose consumption rate and lactic acid concentration of the culture medium in DCA-treated cells were measured using Glucose-GLO and Lactate-GLO kits. (G) WT and KO cells were treated with Ctrl, 10 mM DCA, or 20 mM DCA for 24 h. Intracellular ROS levels were detected using the abcam cellular ROS assay kit and analyzed by flow cytometry. (H) Quantitation of intracellular ROS levels. (I) Cells were treated with Ctrl, 10 mM, 20 mM, or 40 mM DCA for 48 h. Cell viability was analyzed using the CCK-8 assay at OD450. (J) Cells were treated with Ctrl, 10 mM, or 20 mM DCA for 7 days. Cells were stained with crystal violet. Colony numbers were quantified in (K). (L) Illustration of the DCA activated the PDH activity to suppress cell viability in KO cells. P < 0.05 (*), P < 0.01 (**).

Article Snippet: TRAP1 CRISPR/Cas9 plasmid was obtained from Santa Cruz (Dallas, TX, USA). pCMV6 and pCMV6/TRAP1 plasmids were obtained from Origene (Rockville, MD, USA).

Techniques: Western Blot, Staining, Microscopy, Concentration Assay, ROS Assay, Flow Cytometry, Quantitation Assay, CCK-8 Assay, Activity Assay

Journal: Oxidative medicine and cellular longevity

Article Title: Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes.

doi: 10.1155/2020/6431517

Figure Lengend Snippet: Figure 3: Effects of TRAP1 on mitochondrial morphology and dysfunction in NRK-52e cells after high-glucose injury. (a) Mitochondrial morphology was observed by TEM. Arrows indicate mitochondria; scale bar, 500 nm. (b) ATP depletion was measured using firefly luciferase. (c, d) Representative plots of MMP and statistical analyses were determined by flow cytometric analysis of tetramethylrhodamine ethyl ester-labeled NRK-52e cells. (e, f) Representative plots and statistical analysis of intracellular ROS in cells labeled with the fluorescent probe CellROX Deep Red and analyzed by flow cytometry. (g, h) Typical fluorescence photomicrograph and quantitative analysis of mitochondrial superoxide (red: MitoSox; blue: DAPI); scale bar, 50 μm. ROS: reactive oxygen species. The results are presented as the mean ± SEM; n = 3, ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001 for each pair of groups indicated.

Article Snippet: After being blocked in 5% BSA for 1 h, the membranes were probed overnight at 4°C with antibodies against TRAP1 (1 : 1000; Novus Biologicals, USA) and GAPDH (1 : 5000; Proteintech, USA).

Techniques: Luciferase, Labeling, Cytometry

Journal: Oxidative medicine and cellular longevity

Article Title: Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes.

doi: 10.1155/2020/6431517

Figure Lengend Snippet: Figure 4: TRAP1 protects against high-glucose-induced mitochondrial dysfunction via mPTP opening regulation. (a, b) Representative graphs and statistical analyses of mPTP by flow cytometry with the fluorescent probe calcein AM labeling. (c) Mitochondrial ultrastructural damage was observed by TEM. Arrows indicate mitochondria; scale bar, 500 nm. (d, e) Typical graphs of mitochondrial membrane potential and statistical analysis determined with tetramethylrhodamine ethyl ester labeling by flow cytometric analysis at 48 h. (f, g) Representative graphs and statistical analysis of mPTP by flow cytometry. mPTP: mitochondrial permeability transition pore. The results are presented as the mean ± SEM; n = 3, ∗p < 0:05, and ∗∗p < 0:01 for each pair of groups indicated.

Article Snippet: After being blocked in 5% BSA for 1 h, the membranes were probed overnight at 4°C with antibodies against TRAP1 (1 : 1000; Novus Biologicals, USA) and GAPDH (1 : 5000; Proteintech, USA).

Techniques: Cytometry, Labeling, Membrane, Permeability

Journal: Oxidative medicine and cellular longevity

Article Title: Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes.

doi: 10.1155/2020/6431517

Figure Lengend Snippet: Figure 6: Effect of TRAP1 overexpression on biochemical parameters and histopathology in vivo. Rats were treated with an injection of STZ to induce diabetes and AAV 2/9 to overexpress TRAP1. Rats transfected with empty-GFP vectors (AAV-VE) are shown for comparison. Blood and kidney tissues were collected after 12 weeks. (a) Western blot graphs and densitometric analyses of TRAP1 expression in kidneys. GAPDH was used as a control. (b–f) Blood glucose (b), serum FRU (c), serum Ccr (d), urine albumin to creatinine (e), and serum urea (f) levels were analyzed. (g) Representative histology of the renal cortex and outer medulla: H&E staining, PAS for glycogen, Sirius red staining for the detection of fibrosis, and Masson’s trichrome staining for connective tissue; scale bar: 50 μm. The results are presented as the mean ± SEM; n = 4‐6, ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001 for each pair of groups indicated.

Article Snippet: After being blocked in 5% BSA for 1 h, the membranes were probed overnight at 4°C with antibodies against TRAP1 (1 : 1000; Novus Biologicals, USA) and GAPDH (1 : 5000; Proteintech, USA).

Techniques: Over Expression, Histopathology, In Vivo, Injection, Transfection, Comparison, Western Blot, Expressing, Control, Staining

Journal: Oxidative medicine and cellular longevity

Article Title: Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes.

doi: 10.1155/2020/6431517

Figure Lengend Snippet: Figure 7: Effects of TRAP1 on diabetes-induced apoptosis and mitochondrial damage in rat kidney. (a) Representative images of (TUNEL) staining (red: TUNEL; blue: DAPI); scale bar, 100 μm. (b) Quantification of TUNEL-positive cells in fields. (c) Mitochondria of tubular epithelial cells viewed by TEM; scale bar, 1 μm. (d) ATP levels measured by firefly luciferase detection. (e, f) Representative photomicrographs and quantification of ROS staining in rat renal tubules; scale bar, 100 μm. The results are presented as the mean ± SEM; n = 4‐6, ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001 for each pair of groups indicated.

Article Snippet: After being blocked in 5% BSA for 1 h, the membranes were probed overnight at 4°C with antibodies against TRAP1 (1 : 1000; Novus Biologicals, USA) and GAPDH (1 : 5000; Proteintech, USA).

Techniques: TUNEL Assay, Staining, Luciferase

Journal: eLife

Article Title: Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia

doi: 10.7554/eLife.63104

Figure Lengend Snippet: ( A ) OXPHOS kinetics in permeabilized HL-60 cells in the presence of DMSO (vehicle), bongkrekic acid (20 µM; ANT inhibitor) or gamitrinib (1 µM; TRAP1 inhibitor with mitochondria-targeted moiety); n = 3 independent experiments. ( B ) Mitochondrial membrane potential (ΔΨ) in HL-60 isolated mitochondria across a ΔG ATP span, followed by CV inhibition with oligomycin; n = 3 independent experiments. Data are presented as mean ± SEM and analyzed by two-way ANOVA ( A ) or paired t-test ( B ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Transfected construct ( Homo sapien ) , shRNA to TRAP1 , Origene , CAT#: TL300868V , shRNA lentiviral particles packaged from pGFP-C-shLenti vector.

Techniques: Membrane, Isolation, Inhibition

Journal: eLife

Article Title: Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia

doi: 10.7554/eLife.63104

Figure Lengend Snippet: ( A ) Log2 Abundance of TRAP1 in PBMC and leukemia cells. ( B ) Comparison of OXPHOS kinetics in presence of the TRAP1 inhibitor, 17-AAG (15 µM); n = 4 independent cell experiments. Comparison of ( C ) Fractional OXPHOS and ( D ) FCCP Effect in MV-4–11 cells treated with DMSO or 17-AAG (15 µM). ( E ) Comparison of respiratory flux inhibition within MV-4–11 cells across a range of ΔG ATP in the absence and presence of 17-AAG (15 µM). Respiration was stimulated by the addition of FCCP (1 µM), followed by PCR titration to manipulate ΔG ATP . ( F ) Relative abundance of TRAP1 mRNA following shRNA knockdown of TRAP1 in MV-4–11 cells. ( G ) Basal respiration in intact cells. ( H ) OXPHOS kinetics in permeabilized MV-4–11 cells infected with lentivirus encoding shRNA targeted to TRAP1 (TRAP1 KD) or scrambled shRNA (Control). ( I–J ) Fractional OXPHOS and FCCP Effect measured in the presence of vehicle (DMSO), 17-AAG (15 µM), or gamitrinib (1 µM) in control and TRAP1 KD cells. ( K ) OXPHOS kinetics in permeabilized MV-4–11 cells in the presence of DMSO or curcumin (10–20 µM). FCCP Effect is graphed to the right. ( L ) Schematic depicting the presumed mechanism of action of 17-AAG, gamitrinib and curcumin in which the compounds selectively block ATP uptake via the VDAC-ANT axis to restore OXPHOS kinetics. ( M ) Cell proliferation expressed as a percentage of Control. ( N ) Cell viability in MV-4–11 cells infected with lentivirus encoding shRNA targeted to TRAP1 or scrambled shRNA and treated for 24 hr with increasing concentrations of Ara-C. Additional treatments included gamitrinib (1 µM), 17-AAG (15 µM) or curcumin (10–20 µM) either alone or plus Ara-C (20 µM). Data depicted as viability based on the percentage of vehicle using the propidium iodide assay. ( A ) n = 4/6/group; ( B–D ) n = 4 independent experiments; ( E–K ) n = 3 independent experiments; ( M ) n = 8 independent experiments; ( N ) n = 4–6 independent experiments. Data are presented as mean ± SEM analyzed by unpaired t-tests ( F–H, M ) two-way ANOVA ( B, K ), one-way ANOVA ( A, I, J, N ), paired t-tests ( B–E ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 6—source data 1. Raw values for ' ' and ' '.

Article Snippet: Transfected construct ( Homo sapien ) , shRNA to TRAP1 , Origene , CAT#: TL300868V , shRNA lentiviral particles packaged from pGFP-C-shLenti vector.

Techniques: Comparison, Inhibition, Titration, shRNA, Knockdown, Infection, Control, Blocking Assay

Journal: eLife

Article Title: Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia

doi: 10.7554/eLife.63104

Figure Lengend Snippet: ( A ) Comparison of FCCP-stimulated respiration with the addition of 17-AAG (15 µM) in permeabilized MV-4–11 cells; n = 3 independent experiments. ( B–D ) OXPHOS kinetics in permeabilized HL-60 cells in the presence of DMSO or 17-AAG (15 µM); n = 3 independent experiments. ( E–F ) OXPHOS kinetics in permeabilized MV-4–11 or KG-1 cells in the presence of DMSO or 17-AAG (15 µM). Cell were energized with either CI (Pyr/M) or CII linked substrates (S/R); n = 3 independent experiments. ( G ) Schematic of changes in ATP, ADP, PCR, and CR concentrations across a range of ΔG ATP. ( H ) Inhibition of respiration by antimycin A (ANT) across a range of ΔG ATP in permeabilized HL-60 cells; n = 3 independent experiments. ( I–J ) Comparison of respiratory flux inhibition within permeabilized AML cells across a range of ΔG ATP induced by functional TRAP1. Respiration was stimulated by the addition of FCCP (1 µM), followed by PCR titration to manipulate ΔG ATP ; n = 3 independent experiments. ( K ) ETS capacity assay in permeabilized MV-4–11 cells infected with lentivirus encoding shRNA targeted to TRAP1 (TRAP1 KD) or scrambled shRNA (Control); n = 3 independent experiments. ( L ) Comparison of OXPHOS kinetics in the presence of DMSO, 17-AAG (15 µM), or gamitrinib (1 µM) in Control and TRAP1 KD cells. Data depicted as a percentage of basal respiration based on oxygen consumption rates obtained prior to digitonin permeabilization; n = 3 independent experiments. Data are presented as mean ± SEM and analyzed by paired t-tests ( A–F, H–J ) or unpaired t-tests ( K–L ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Transfected construct ( Homo sapien ) , shRNA to TRAP1 , Origene , CAT#: TL300868V , shRNA lentiviral particles packaged from pGFP-C-shLenti vector.

Techniques: Comparison, Inhibition, Functional Assay, Titration, Infection, shRNA, Control

Journal: eLife

Article Title: Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia

doi: 10.7554/eLife.63104

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo sapien ) , shRNA to TRAP1 , Origene , CAT#: TL300868V , shRNA lentiviral particles packaged from pGFP-C-shLenti vector.

Techniques: Isolation, Lysis, Transfection, Construct, shRNA, Plasmid Preparation, Sequencing, Recombinant, Reverse Transcription, Software