Journal: bioRxiv

Article Title: Glutathionylation of Pyruvate Dehydrogenase Complex E2 Protein During Acute Inflammation Is Magnified By Mitochondrial Oxidative Stress, Enhancing Cell Death

doi: 10.1101/2023.01.26.525791

Figure Lengend Snippet: (A) Three components (E1, E2 and E3) of PDC together catalyze the decarboxylation of pyruvate to produce acetyl-CoA. (B) PDCE3 under permissive conditions exhibits NADH-dependent oxidase activity, producing superoxide and additional radical reactions. (C) Biotinylated glutathione (GSH) was used to detect glutathionylation of PDCE2 in THP-1 pro-monocytes after treatment with 1 μg/mL LPS. After affinity capture using streptavidin beads and elution with dithiothreitol, PDCE2 was visualized by immunoblotting (n=5). A significant increase in glutathionylated PDCE2 was seen at 6 h and 24 h LPS treatment (* p<0.05). (D) Glutathionylated PDCE2 generated in human primary monocytes after treatment with 100 ng/mL LPS was assessed as in A (n=3) and showed a significant increase in glutathionylated PDCE2 at 24 h (** p<0.01). (E) A PDCE2 expression plasmid including sequences encoding an N-terminal FLAG tag was designed to replace lipoylated lysine residues (K132 and K259) with arginine residues, enabling expression of wild type (WT) and non-lipoylated PDCE2. (F) Using CRISPR-generated PDCE2 knockout THP-1 cells as host, plasmid-transfected cells expressing WT or K132R/K259R PDCE2 were treated with LPS or buffer for 6 h, followed by immunoprecipitation (IP) of the lysates for the FLAG-tagged proteins and immunoblotting for biotin to detect glutathionylation (Bio-GEE incorporation) (n=5). The arrow indicates the band expected for PDCE2.

Article Snippet: A PDCE2 expression plasmid from SinoBiological (HG15002-UT) served as template for insertion of an N-terminal FLAG tag sequence and for generating a double mutant substituting Arg for two Lys residues (at 132 and 259).

Techniques: Activity Assay, Western Blot, Generated, Expressing, Plasmid Preparation, FLAG-tag, CRISPR, Knock-Out, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: Glutathionylation of Pyruvate Dehydrogenase Complex E2 Protein During Acute Inflammation Is Magnified By Mitochondrial Oxidative Stress, Enhancing Cell Death

doi: 10.1101/2023.01.26.525791

Figure Lengend Snippet: (A) Mitochondrial ROS production was assessed in LPS-treated wild type (WT) THP-1 cells in comparison with CRISPR-mediated PDCE2 knockout (KO) THP-1 cells using 5 μM MitoSox Red assessed 6 h after LPS or buffer addition. The strong ROS response to LPS exhibited by WT THP-1 cells was blunted in PDCE2 KO cells (n=8). (B) Mitochondrial H 2 O 2 production was assessed in THP-1 WT and KO cells similar to (A), but using MitoPY1. A significant increase in mitochondrial H 2 O 2 production was observed in response to 6 h LPS treatment of WT cells, but blunted in PDCE2 KO cells (n=8). (C) Cell death was assessed in WT and PDCE2 KO THP-1 cells using Promega Multi-Tox Fluor assay. A significant increase in Dead Cell/Live Cell fluorescence ratio was observed in response to 6 h LPS treatment of WT cells, but not in PDCE2 KO cells (n=13). (D) LDH release as a measure of cell death was assessed in WT and PDCE2 KO THP-1 cells using Promega Homogenous Membrane Integrity assay. A significant increase in percent cell death was observed in response to overnight LPS treatment of WT cells but not PDCE2 KO cells (n=32). (A-D) All statistical analyses were conducted using one-way ANOVA with Tukey’s multiple comparisons post-test; * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: A PDCE2 expression plasmid from SinoBiological (HG15002-UT) served as template for insertion of an N-terminal FLAG tag sequence and for generating a double mutant substituting Arg for two Lys residues (at 132 and 259).

Techniques: CRISPR, Knock-Out, Fluorescence, Integrity Assay

Journal: bioRxiv

Article Title: Glutathionylation of Pyruvate Dehydrogenase Complex E2 Protein During Acute Inflammation Is Magnified By Mitochondrial Oxidative Stress, Enhancing Cell Death

doi: 10.1101/2023.01.26.525791

Figure Lengend Snippet: (A) Mitochondrial ROS production was assessed in PDCE2 KO THP-1 cells transfected with Empty Vector or PDCE2 expression plasmids with and without LPS stimulation using MitoSox Red (n=12, * p<0.05, n.s. = not significant). (B) Cell death was assessed in transfected PDCE2 KO THP-1 cells as in (A), in this case using Promega Multi-Tox Fluor assay (n=14). (A and B) Statistically-significant increases in mitochondrial ROS production and cell death observed upon treatment of WT-expressing cells with LPS were not observed in the cells expressing PDCE2 lacking the lipoyl-linking Lys residues (K132R/K259R).

Article Snippet: A PDCE2 expression plasmid from SinoBiological (HG15002-UT) served as template for insertion of an N-terminal FLAG tag sequence and for generating a double mutant substituting Arg for two Lys residues (at 132 and 259).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Glutathionylation of Pyruvate Dehydrogenase Complex E2 Protein During Acute Inflammation Is Magnified By Mitochondrial Oxidative Stress, Enhancing Cell Death

doi: 10.1101/2023.01.26.525791

Figure Lengend Snippet: (A) BioGEE incorporation assays as conducted in were used to assess glutathionylation of PDCE2 in THP-1 promonocytes after 6 h treatment with 10 μM MitoCDNB (MCDNB), 1 μg/mL LPS, or a combination of the two (n=5). A significant increase in glutathionylated PDCE2 was seen with the combined treatments relative to either treatment alone (* p<0.05). (B) MitoSox Red was used to assess ROS in WT and PDCE2 KO THP-1 promonocytes. A significant decrease in cell death was seen with the combined treatments in PDCE2 CRISPR cells relative to their Wild Type Counterparts (n=23). (C) Promega Multi-Tox Fluor assay was used to assess cell death in in WT and PDCE2 KO THP-1 cells. The increase in cell death observed with the combined treatments in WT THP-1 cells was significantly less in PDCE2 KO cells (n=12). (D) LDH release was assessed as an independent measure of cell death using Promega Homogenous Membrane Integrity assay. Again the LPS-induced increase in LDH release observed with the combined treatments for WT cells was greater that for PDCE2 KO cells (n=24). (A-D) , * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: A PDCE2 expression plasmid from SinoBiological (HG15002-UT) served as template for insertion of an N-terminal FLAG tag sequence and for generating a double mutant substituting Arg for two Lys residues (at 132 and 259).

Techniques: CRISPR, Integrity Assay

Journal: Biomaterials

Article Title: Immunogenic cuproptosis in cancer immunotherapy via an in situ cuproptosis-inducing system.

doi: 10.1016/j.biomaterials.2025.123201

Figure Lengend Snippet: Fig. 2. Cuproptosis in CRC cells leaded to ICD (A-D) Western blotting analysis of the oligomerization of lipoylated DLAT, LIAS and FDX1 in MC38 (A), HCT116 (B), SW480 (C) and SW620 (D) cells after different treatments for 24h. (E–H) Western blotting analysis of HSP-70 and HSP-90 expression in HCT116 (E–F) and MC38 (G–H) cells after the different treatment for 12h and 24h. (I–J) Extracellular levels of ATP (I) and HMGB1 (J) after different treatments were shown in each bar. (K) The transposition of CRT from ER and the exposure of CRT on the cytomembrane when cuproptosis happened was observed in HCT116 cells. ER-tracker means the endoplasmic reticulum probe and Dil means the cytomembrane probe. Scale bar = 10 μm. (L) Flow chart of the co-culture experiments. (M) CD11c and CD86 positive cells rates of dendritic cells co-cultured with MC38 cells after different treatments were shown in each bar. (N) CD80 and CD86 positive cells rates of RAW264.7 cells co-cultured with MC38 cells after different treatments were shown in each bar. (O) CD11c and CD86 positive cells rates of dendritic cells co-cultured with MC38 cells after treatment with different doses of Imiquimod were shown in each bar. (N) CD80 and CD86 positive cells rates of RAW264.7 cells co-cultured with MC38 cells after treatment with different doses of Imiquimod were shown in each bar. (Q) Transcriptional levels of the DC-secreted pro-inflammatory cytokines, anti-inflammatory cytokines and pro-inflammatory chemokines were analyzed by qRT-PCR for the co-cultured DCs. (R) Extracellular levels of IL-18 of the co-cultured DCs analyzed by ELISA were shown in each bar. (S) Extracellular levels of IL-18 of the co-cultured DCs analyzed by ELISA were shown in each bar. For Imiquimod, +means 5 μg/mL Imiquimod, ++ means 10 μg/mL Imiquimod and +++ means 20 μg/mL Imiquimod. Data are present as means ± SEM (n ≥3). Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs the control group.

Article Snippet: Antibodies against LIAS, Cleaved Caspase3, Phospho-MLKL (Ser358), CD8a, CRT, HSP-70, HSP-90, Ki67, DLAT, CHOP, EIF2A, PERK, GADD34, iNOS, and β-actin were sourced from Proteintech (Wuhan, China), and antibodies against CD4, CD11c, F4/80, CD86, and CD206 were obtained from CUSABIO (Houston, USA).

Techniques: Western Blot, Expressing, Co-Culture Assay, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Biomaterials

Article Title: Immunogenic cuproptosis in cancer immunotherapy via an in situ cuproptosis-inducing system.

doi: 10.1016/j.biomaterials.2025.123201

Figure Lengend Snippet: Fig. 4. Preparation and characterization of the Cu2+-hydrogel (A) The structure of 4-ArmPEG-DA molecule. (B) The three-dimensional network structure of the hydrogel. (C) Photographs showing that after the two solutions were mixed together at room temperature, they could change from liquid to solid within a few minutes in vitro. (D) Microstructure analysis of the hydrogel and drug-coated variant was conducted via cryo-scanning electron microscopy. Scale bar = 500 μm. (E) Rheology properties of the formation of the 10 wt%, 20 wt% and 30 wt% hydrogels. G′ means storage modulus and G″ means loss modulus. (F) Rheology properties of the formation of hydrogels with pH value = 10.0, 11.8 and 13.0. G′ means storage modulus and G″ means loss modulus. (G) Rheology properties of the toughness and stability of 20 wt% hydrogel with pH value = 11.8. G′ means storage modulus and G″ means loss modulus. (H) Swelling ratios of hydrogels were measured at different time points of 1 d, 2 d, 3 d, 4 d, 6 d, 8 d, 10 d, 13 d, 16 d, 21 d, 31 d, 37 d, 43 d, 63 d, 81 d, 90 d and 97 d after immersing in the PBS. (I) CCK-8 assay analyzed the cytotoxicity of 20 wt% hydrogel with pH value = 11.8 after treatment for 0 h, 24 h, 48 h, 72 h and 96 h to evaluate its cytocompatibility in MC38 cells. (J) Cumulative release profiles of Cu2+, Elesclomol and Imiquimod from the hydrogel incubated in PBS at the time points of 1 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, 3 d, 4 d, 6d, 8 d and 10 d. (K) In vivo release profiles analysis by coating the hydrogel with a hydrophilic dye, IR820, for subcutaneous injection in mice at 0, 1, 3, 7, 14 and 30 days after injection. (L) Cell viability was detected by CCK-8 assay after administration of different treatments for 24 h to show the cell viability inhibited effects caused by the hydrogel delivery system in HCT116, MC38, SW480 and SW620 cells. (M–P) Western blotting analysis of DLAT, HSP-70, HSP-90, CRT, GADD34, CHOP, EIF2A and PERK expression in MC38 (M), HCT116 (N), SW480 (O) and SW620 (P) cells after different treatments. (Q) The migration area of the wound healing assay of SW480 and HCT116 cells was calculated by Image J. Data are presented as means ± SEM (n ≥3).

Article Snippet: Antibodies against LIAS, Cleaved Caspase3, Phospho-MLKL (Ser358), CD8a, CRT, HSP-70, HSP-90, Ki67, DLAT, CHOP, EIF2A, PERK, GADD34, iNOS, and β-actin were sourced from Proteintech (Wuhan, China), and antibodies against CD4, CD11c, F4/80, CD86, and CD206 were obtained from CUSABIO (Houston, USA).

Techniques: In Vitro, Variant Assay, Electron Microscopy, CCK-8 Assay, Incubation, In Vivo, Injection, Western Blot, Expressing, Migration, Wound Healing Assay

Journal: Biomaterials

Article Title: Immunogenic cuproptosis in cancer immunotherapy via an in situ cuproptosis-inducing system.

doi: 10.1016/j.biomaterials.2025.123201

Figure Lengend Snippet: Fig. 5. Anti-tumor effects induced by the cuproptosis-based immunotherapy in vivo (A) The flow chart of the animal experiment. (B) In vivo image of the tumors at 0, 3, 7 and 14 days after injection. (C–D) Luminescence of the primary (C) and distant (D) tumors during the process was counted. (E–F) Tumor weight for primary tumors (E) and distant tumors (F). (G) Body weight of the mice after the treatments. (H) Western blotting analysis of HSP-70, HSP-90 and the oligomerization of lipoylated DLAT in the primary tumor tissues after the different treatment in vivo. Data are present as means ± SEM (n = 5). Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs the control group.

Article Snippet: Antibodies against LIAS, Cleaved Caspase3, Phospho-MLKL (Ser358), CD8a, CRT, HSP-70, HSP-90, Ki67, DLAT, CHOP, EIF2A, PERK, GADD34, iNOS, and β-actin were sourced from Proteintech (Wuhan, China), and antibodies against CD4, CD11c, F4/80, CD86, and CD206 were obtained from CUSABIO (Houston, USA).

Techniques: In Vivo, Injection, Western Blot, Control

Journal: Biomolecules

Article Title: Copper Chelation by Penicillamine Protects Against Doxorubicin-Induced Cardiomyopathy by Suppressing FDX1-Mediated Cuproptosis

doi: 10.3390/biom15091320

Figure Lengend Snippet: Two-dimensional and 3D interaction diagrams represent the key for the types of interaction between controls and selected protein receptors: ( A ) FDX1 (ferredoxin 1) 3P1M protein, ( C ) SLC31A13 2LS2 protein, ( E ) DLAT (dihydrolipoamide acetyltransferase) 3B8K protein. penicillamine and selected protein receptors: ( B ) FDX1 (ferredoxin 1) 3P1M protein, ( D ) SLC31A13 2LS2 protein, ( F ) DLAT (dihydrolipoamide acetyltransferase) 3B8K protein.

Article Snippet: 3-μm-thickness paraffin sections were prepared and used for HE staining (for examination of the morphological changes); MT staining (for analysis of collagen and extracellular matrix “ECM” deposition); and immunohistochemical staining for the cuproptosis dynamic protein markers (SLC31A1, FDX1, and DLAT), using anti-SLC31A1/CTR1 antibody (Cat. No. GTX48534, GeneTex, Irvine, CA, USA); Anti FDX1 Polyclonal antibody (Cat. No. 12592-1-AP, Proteintech, Chicago, IL, USA); and anti-DLAT antibody (Cat. No. CSB- PA445587 , CUSABIO, Houston, TX, USA), respectively ( ).

Techniques: