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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, 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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, 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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, 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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, 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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, 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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, 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 inhibitor (G-TPP) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, USA).

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