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trap1 polyclonal antibody  (Proteintech)


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    Proteintech trap1 polyclonal antibody
    In the retina of STZ-induced diabetic retinopathy (DR) rat models, the level of <t>TRAP1</t> decreases. (A) Changes in body weight during the modeling period of diabetic and control rats. (n=8) ( B ) FBG levels during the modeling period of diabetic and control rats. (n=8) ( C ) Representative micrographs of retinal sections stained with H&E from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Scale bar=50μm. (n=3) ( D ) Western blot analysis of total TRAP1 in retinal tissues from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Quantitative data are shown in the right panel. (n=3) ( E ) Total RNA was extracted from retinal tissues of control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups, and TRAP1 mRNA expression was detected by qRT-PCR. (n=3) ( F ) Immunofluorescence staining of retinal tissue sections from each group of rats using anti-TRAP1 antibody (yellow) and DAPI (blue) for nuclear staining. The error bars in the above histograms represent the mean±SD of independent experiments.*P<0.05, **P<0.01, ***P<0.001,****P<0.0001.
    Trap1 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trap1 polyclonal antibody/product/Proteintech
    Average 93 stars, based on 22 article reviews
    trap1 polyclonal antibody - by Bioz Stars, 2026-02
    93/100 stars

    Images

    1) Product Images from "TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function"

    Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    doi: 10.2147/OPTH.S521660

    In the retina of STZ-induced diabetic retinopathy (DR) rat models, the level of TRAP1 decreases. (A) Changes in body weight during the modeling period of diabetic and control rats. (n=8) ( B ) FBG levels during the modeling period of diabetic and control rats. (n=8) ( C ) Representative micrographs of retinal sections stained with H&E from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Scale bar=50μm. (n=3) ( D ) Western blot analysis of total TRAP1 in retinal tissues from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Quantitative data are shown in the right panel. (n=3) ( E ) Total RNA was extracted from retinal tissues of control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups, and TRAP1 mRNA expression was detected by qRT-PCR. (n=3) ( F ) Immunofluorescence staining of retinal tissue sections from each group of rats using anti-TRAP1 antibody (yellow) and DAPI (blue) for nuclear staining. The error bars in the above histograms represent the mean±SD of independent experiments.*P<0.05, **P<0.01, ***P<0.001,****P<0.0001.
    Figure Legend Snippet: In the retina of STZ-induced diabetic retinopathy (DR) rat models, the level of TRAP1 decreases. (A) Changes in body weight during the modeling period of diabetic and control rats. (n=8) ( B ) FBG levels during the modeling period of diabetic and control rats. (n=8) ( C ) Representative micrographs of retinal sections stained with H&E from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Scale bar=50μm. (n=3) ( D ) Western blot analysis of total TRAP1 in retinal tissues from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Quantitative data are shown in the right panel. (n=3) ( E ) Total RNA was extracted from retinal tissues of control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups, and TRAP1 mRNA expression was detected by qRT-PCR. (n=3) ( F ) Immunofluorescence staining of retinal tissue sections from each group of rats using anti-TRAP1 antibody (yellow) and DAPI (blue) for nuclear staining. The error bars in the above histograms represent the mean±SD of independent experiments.*P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Techniques Used: Control, Staining, Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence

    HG promotes mitochondrial dysfunction in ARPE-19 cells, accompanied by a decrease in TRAP1 levels. ( A ) Cell viability of ARPE-19 cells cultured with different glucose concentrations for 4 days. (n=4) ( B ) Cell viability of ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=4) ( C ) Intracellular ROS levels in cells cultured with 50mM glucose for 1–6 days. (n=3) ( D ) Identification of Δψ m through JC-1 staining after culturing ARPE-19 cells with 50mM glucose for 6 days. Images captured under fluorescence microscope show red fluorescence representing polymer form, indicating intact Δψ m, and green fluorescence representing monomer form, indicating decreased Δψ m. Quantified data presented on the right. Scale bar=20μm. (n=3) ( E ) ARPE-19 cells cultured with 50mM glucose for 6 days and treated with Calcein AM (1X) and CoCl2 (1X). Images captured under fluorescence microscope shown on the left. Quantified data presented on the right. Scale bar=20μm. (n=3) ( F ) TEM images of mitochondrial ultrastructure in ARPE-19 cells cultured with 50mM glucose for 6 days and control ARPE-19 cells. Quantified data presented on the right. Scale bar=2μm. ( G ) Western blot analysis of TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( H ) Western blot analysis of intramitochondrial TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days after mitochondrial extraction. (n=3) ( I ) Quantified data corresponding to Figure ( G ). ( J ) qRT-PCR analysis of TRAP1 mRNA expression in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( K ) Quantified data corresponding to Figure ( H ). The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.
    Figure Legend Snippet: HG promotes mitochondrial dysfunction in ARPE-19 cells, accompanied by a decrease in TRAP1 levels. ( A ) Cell viability of ARPE-19 cells cultured with different glucose concentrations for 4 days. (n=4) ( B ) Cell viability of ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=4) ( C ) Intracellular ROS levels in cells cultured with 50mM glucose for 1–6 days. (n=3) ( D ) Identification of Δψ m through JC-1 staining after culturing ARPE-19 cells with 50mM glucose for 6 days. Images captured under fluorescence microscope show red fluorescence representing polymer form, indicating intact Δψ m, and green fluorescence representing monomer form, indicating decreased Δψ m. Quantified data presented on the right. Scale bar=20μm. (n=3) ( E ) ARPE-19 cells cultured with 50mM glucose for 6 days and treated with Calcein AM (1X) and CoCl2 (1X). Images captured under fluorescence microscope shown on the left. Quantified data presented on the right. Scale bar=20μm. (n=3) ( F ) TEM images of mitochondrial ultrastructure in ARPE-19 cells cultured with 50mM glucose for 6 days and control ARPE-19 cells. Quantified data presented on the right. Scale bar=2μm. ( G ) Western blot analysis of TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( H ) Western blot analysis of intramitochondrial TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days after mitochondrial extraction. (n=3) ( I ) Quantified data corresponding to Figure ( G ). ( J ) qRT-PCR analysis of TRAP1 mRNA expression in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( K ) Quantified data corresponding to Figure ( H ). The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Techniques Used: Cell Culture, Staining, Fluorescence, Microscopy, Polymer, Control, Western Blot, Extraction, Quantitative RT-PCR, Expressing

    TRAP1 rescues mitochondrial damage under high-glucose stimulation. ( A ) The left panel depicts Western blot analysis of TRAP1 overexpression and knockdown cell lines. The right panel shows the quantified data. (n=3) ( B ) Total RNA was extracted from ARPE-19 cells overexpressing or knockdown for TRAP1, followed by qRT-PCR to assess TRAP1 mRNA expression. (n=3) ( C ) Immunofluorescence staining of TRAP1 (red) and Tom20 (green) demonstrates their colocalization in TRAP1 OE and shTRAP1 cells. Cell nuclei were stained with DAPI (blue). Scale bar = 20μm. (n=3) ( D )The upper panel illustrates cell viability of NC group, cells transduced with overexpression empty virus, and cells transduced with knockdown empty virus cultured for 6 days under 50mM glucose and control conditions. The lower panel represents cell viability of NC, TRAP1 OE , and shTRAP1 cells cultured for 6 days under 50mM glucose and control conditions. (n=4) ( E ) Intracellular ROS levels were measured after 6 days of incubation with 50mM glucose. (n=3) ( F ) Cells cultured with 50mM glucose for 6 days were subjected to JC-1 staining to assess Δψ m. Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( G ) Quantified data from panel ( F ) are presented. ( H ) Cells cultured with 50mM glucose for 6 days were treated with Calcein AM (1X) and CoCl2 (1x). Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( I ) Quantified data from panel ( H ) are presented. ( J ) TEM was performed to evaluate mitochondrial ultrastructure after 6 days of incubation with 50mM glucose. Scale bar = 2μm. ( K ) Quantified data from panel ( J ) are presented. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.
    Figure Legend Snippet: TRAP1 rescues mitochondrial damage under high-glucose stimulation. ( A ) The left panel depicts Western blot analysis of TRAP1 overexpression and knockdown cell lines. The right panel shows the quantified data. (n=3) ( B ) Total RNA was extracted from ARPE-19 cells overexpressing or knockdown for TRAP1, followed by qRT-PCR to assess TRAP1 mRNA expression. (n=3) ( C ) Immunofluorescence staining of TRAP1 (red) and Tom20 (green) demonstrates their colocalization in TRAP1 OE and shTRAP1 cells. Cell nuclei were stained with DAPI (blue). Scale bar = 20μm. (n=3) ( D )The upper panel illustrates cell viability of NC group, cells transduced with overexpression empty virus, and cells transduced with knockdown empty virus cultured for 6 days under 50mM glucose and control conditions. The lower panel represents cell viability of NC, TRAP1 OE , and shTRAP1 cells cultured for 6 days under 50mM glucose and control conditions. (n=4) ( E ) Intracellular ROS levels were measured after 6 days of incubation with 50mM glucose. (n=3) ( F ) Cells cultured with 50mM glucose for 6 days were subjected to JC-1 staining to assess Δψ m. Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( G ) Quantified data from panel ( F ) are presented. ( H ) Cells cultured with 50mM glucose for 6 days were treated with Calcein AM (1X) and CoCl2 (1x). Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( I ) Quantified data from panel ( H ) are presented. ( J ) TEM was performed to evaluate mitochondrial ultrastructure after 6 days of incubation with 50mM glucose. Scale bar = 2μm. ( K ) Quantified data from panel ( J ) are presented. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Techniques Used: Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Transduction, Virus, Cell Culture, Control, Incubation, Fluorescence, Microscopy

    Knocking down TRAP1 affects oxidative stress and related mitochondrial functions in ARPE-19 cells. ( A ) Volcano plot of differentially expressed genes (DEGs) between shTRAP1 and NC, with yellow representing significantly upregulated genes and blue representing significantly downregulated genes (|log2 fold change|>1, log10 adjusted p-values<0.05). ( B ) Heatmap of DEGs between shTRAP1 and NC. ( C ) Venn diagram showing a high overlap between DEGs in the shTRAP1 group and oxidative stress-related genes (OS) and diabetic retinopathy-related risk genes (DR). ( D ) Gene Ontology (GO) enrichment analysis of DEGs. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs.
    Figure Legend Snippet: Knocking down TRAP1 affects oxidative stress and related mitochondrial functions in ARPE-19 cells. ( A ) Volcano plot of differentially expressed genes (DEGs) between shTRAP1 and NC, with yellow representing significantly upregulated genes and blue representing significantly downregulated genes (|log2 fold change|>1, log10 adjusted p-values<0.05). ( B ) Heatmap of DEGs between shTRAP1 and NC. ( C ) Venn diagram showing a high overlap between DEGs in the shTRAP1 group and oxidative stress-related genes (OS) and diabetic retinopathy-related risk genes (DR). ( D ) Gene Ontology (GO) enrichment analysis of DEGs. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs.

    Techniques Used:

    TRAP1 alleviates high glucose-induced cellular damage by inhibiting mitochondrial ferroptosis.( A ) Activity of shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1, Z-VAD, 3-MA, and Nec-1. (n=3) ( B ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=4) ( C ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( D ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( E ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( F ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. R-BODIPY: Reduced BODIPY(red). O-BODIPY: Oxidized BODIPY(green). Right panel: Quantitative data. Scale bar=20μm. (n=3) ( G ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=4) ( H ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( I ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( J ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( K ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. Right panel: Quantitative data. Scale bar= 20μm. (n=3) ( L ) In Control, OE, and OE+HG groups of ARPE19 cells, immunoprecipitation (IP) was performed using TRAP1 antibody, and the enriched proteins were analyzed by Western Blot with antibodies against ACSL1, ACSL4, and CYB5R1. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.
    Figure Legend Snippet: TRAP1 alleviates high glucose-induced cellular damage by inhibiting mitochondrial ferroptosis.( A ) Activity of shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1, Z-VAD, 3-MA, and Nec-1. (n=3) ( B ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=4) ( C ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( D ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( E ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( F ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. R-BODIPY: Reduced BODIPY(red). O-BODIPY: Oxidized BODIPY(green). Right panel: Quantitative data. Scale bar=20μm. (n=3) ( G ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=4) ( H ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( I ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( J ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( K ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. Right panel: Quantitative data. Scale bar= 20μm. (n=3) ( L ) In Control, OE, and OE+HG groups of ARPE19 cells, immunoprecipitation (IP) was performed using TRAP1 antibody, and the enriched proteins were analyzed by Western Blot with antibodies against ACSL1, ACSL4, and CYB5R1. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Techniques Used: Activity Assay, Concentration Assay, Fluorescence, Microscopy, Control, Immunoprecipitation, Western Blot



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    90
    GeneTex trap1 polyclonal rabbit antibody
    OMM severing, IMM ubiquitination and distribution of <t>TRAP1</t> and CPOX in Acn-treated HCT116 and HeLa cells. (A–C) Examples of mitochondria with severed OMM from WT HCT116 (A and B) and WT HeLa (C) cells treated with Acn for 6 h and immunostained to detect conjugated Ub (FK2; green in A–C), Tom20 (blue in A–C), Tom40 (red in A), Fis1 (red in B), and SDHA (red in C). Scale bars represent 1 µm. (D) Quantification of circular mitochondrial ubiquitination in WT HeLa cells treated as indicated in the figure. Data are represented by mean ± SD; n = 3 ( n = 60 cells per experiment). *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal-Wallis with Dunn post-hoc analysis (α = 0.05). (E) Total cell lysates obtained from HeLa (cervical adenocarcinoma), HCT116 (colorectal carcinoma), H4 (neuroglioma), HepG2 (hepatocellular carcinoma), SH-SY5Y (neuroblastoma), U251 (glioblastoma), A549 (lung epithelial carcinoma), and HUH7 (hepatocellular carcinoma) were subjected to Western blot to detect Parkin. The upper “Parkin” panel show blots detected with a standard ECL detection reagent (Pico), and the Parkin panel second from the top was detected using a more sensitive ECL detection reagent (Femto). Rat brain lysate was included as a reference. Tom20 was used as loading control. Numbers in the bottom panel indicate expression of Parkin in all analyzed cells relative to SH-SY5Y neuroblastoma cells that showed the highest expression of this protein. The values were normalized to Tom20 levels. (F–K) Distribution and expression of TRAP1 and CPOX was analyzed in WT and Drp1 −/− HCT116 and WT HeLa cells. WT (F) and Drp1 −/− HCT116 (G and H) cells and WT HeLa cells (I and J) were treated with DMSO (G and I) or Acn (F, H, and J) for 6 h followed by immunostaining to detect cytochrome c (Cyt. c; green), Tom20 (blue), TRAP1 (red in F), and CPOX (red in G and H). Detail images are from areas marked with white rectangles. Scale bars represent 20 µm in F–J; 2 µm in detail images in F, G, and H; and 5 µm in detail images in I and J. Arrowheads in F indicate mitochondria with high cytochrome c/low TRAP1 (red), high TRAP1/low cytochrome c (yellow), and high cytochrome c/high TRAP1 (blue). (K) Expression levels of CPOX, TRAP1, and cytochrome c in WT HeLa cells and WT, ATG5 −/− , and Drp1 −/− HCT116 cells was analyzed by Western blot. Tom20 was used as a loading control. Two exposures of CPOX blot are shown.
    Trap1 Polyclonal Rabbit Antibody, supplied by GeneTex, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    In the retina of STZ-induced diabetic retinopathy (DR) rat models, the level of TRAP1 decreases. (A) Changes in body weight during the modeling period of diabetic and control rats. (n=8) ( B ) FBG levels during the modeling period of diabetic and control rats. (n=8) ( C ) Representative micrographs of retinal sections stained with H&E from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Scale bar=50μm. (n=3) ( D ) Western blot analysis of total TRAP1 in retinal tissues from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Quantitative data are shown in the right panel. (n=3) ( E ) Total RNA was extracted from retinal tissues of control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups, and TRAP1 mRNA expression was detected by qRT-PCR. (n=3) ( F ) Immunofluorescence staining of retinal tissue sections from each group of rats using anti-TRAP1 antibody (yellow) and DAPI (blue) for nuclear staining. The error bars in the above histograms represent the mean±SD of independent experiments.*P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

    doi: 10.2147/OPTH.S521660

    Figure Lengend Snippet: In the retina of STZ-induced diabetic retinopathy (DR) rat models, the level of TRAP1 decreases. (A) Changes in body weight during the modeling period of diabetic and control rats. (n=8) ( B ) FBG levels during the modeling period of diabetic and control rats. (n=8) ( C ) Representative micrographs of retinal sections stained with H&E from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Scale bar=50μm. (n=3) ( D ) Western blot analysis of total TRAP1 in retinal tissues from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Quantitative data are shown in the right panel. (n=3) ( E ) Total RNA was extracted from retinal tissues of control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups, and TRAP1 mRNA expression was detected by qRT-PCR. (n=3) ( F ) Immunofluorescence staining of retinal tissue sections from each group of rats using anti-TRAP1 antibody (yellow) and DAPI (blue) for nuclear staining. The error bars in the above histograms represent the mean±SD of independent experiments.*P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

    Techniques: Control, Staining, Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence

    HG promotes mitochondrial dysfunction in ARPE-19 cells, accompanied by a decrease in TRAP1 levels. ( A ) Cell viability of ARPE-19 cells cultured with different glucose concentrations for 4 days. (n=4) ( B ) Cell viability of ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=4) ( C ) Intracellular ROS levels in cells cultured with 50mM glucose for 1–6 days. (n=3) ( D ) Identification of Δψ m through JC-1 staining after culturing ARPE-19 cells with 50mM glucose for 6 days. Images captured under fluorescence microscope show red fluorescence representing polymer form, indicating intact Δψ m, and green fluorescence representing monomer form, indicating decreased Δψ m. Quantified data presented on the right. Scale bar=20μm. (n=3) ( E ) ARPE-19 cells cultured with 50mM glucose for 6 days and treated with Calcein AM (1X) and CoCl2 (1X). Images captured under fluorescence microscope shown on the left. Quantified data presented on the right. Scale bar=20μm. (n=3) ( F ) TEM images of mitochondrial ultrastructure in ARPE-19 cells cultured with 50mM glucose for 6 days and control ARPE-19 cells. Quantified data presented on the right. Scale bar=2μm. ( G ) Western blot analysis of TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( H ) Western blot analysis of intramitochondrial TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days after mitochondrial extraction. (n=3) ( I ) Quantified data corresponding to Figure ( G ). ( J ) qRT-PCR analysis of TRAP1 mRNA expression in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( K ) Quantified data corresponding to Figure ( H ). The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

    doi: 10.2147/OPTH.S521660

    Figure Lengend Snippet: HG promotes mitochondrial dysfunction in ARPE-19 cells, accompanied by a decrease in TRAP1 levels. ( A ) Cell viability of ARPE-19 cells cultured with different glucose concentrations for 4 days. (n=4) ( B ) Cell viability of ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=4) ( C ) Intracellular ROS levels in cells cultured with 50mM glucose for 1–6 days. (n=3) ( D ) Identification of Δψ m through JC-1 staining after culturing ARPE-19 cells with 50mM glucose for 6 days. Images captured under fluorescence microscope show red fluorescence representing polymer form, indicating intact Δψ m, and green fluorescence representing monomer form, indicating decreased Δψ m. Quantified data presented on the right. Scale bar=20μm. (n=3) ( E ) ARPE-19 cells cultured with 50mM glucose for 6 days and treated with Calcein AM (1X) and CoCl2 (1X). Images captured under fluorescence microscope shown on the left. Quantified data presented on the right. Scale bar=20μm. (n=3) ( F ) TEM images of mitochondrial ultrastructure in ARPE-19 cells cultured with 50mM glucose for 6 days and control ARPE-19 cells. Quantified data presented on the right. Scale bar=2μm. ( G ) Western blot analysis of TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( H ) Western blot analysis of intramitochondrial TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days after mitochondrial extraction. (n=3) ( I ) Quantified data corresponding to Figure ( G ). ( J ) qRT-PCR analysis of TRAP1 mRNA expression in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( K ) Quantified data corresponding to Figure ( H ). The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

    Techniques: Cell Culture, Staining, Fluorescence, Microscopy, Polymer, Control, Western Blot, Extraction, Quantitative RT-PCR, Expressing

    TRAP1 rescues mitochondrial damage under high-glucose stimulation. ( A ) The left panel depicts Western blot analysis of TRAP1 overexpression and knockdown cell lines. The right panel shows the quantified data. (n=3) ( B ) Total RNA was extracted from ARPE-19 cells overexpressing or knockdown for TRAP1, followed by qRT-PCR to assess TRAP1 mRNA expression. (n=3) ( C ) Immunofluorescence staining of TRAP1 (red) and Tom20 (green) demonstrates their colocalization in TRAP1 OE and shTRAP1 cells. Cell nuclei were stained with DAPI (blue). Scale bar = 20μm. (n=3) ( D )The upper panel illustrates cell viability of NC group, cells transduced with overexpression empty virus, and cells transduced with knockdown empty virus cultured for 6 days under 50mM glucose and control conditions. The lower panel represents cell viability of NC, TRAP1 OE , and shTRAP1 cells cultured for 6 days under 50mM glucose and control conditions. (n=4) ( E ) Intracellular ROS levels were measured after 6 days of incubation with 50mM glucose. (n=3) ( F ) Cells cultured with 50mM glucose for 6 days were subjected to JC-1 staining to assess Δψ m. Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( G ) Quantified data from panel ( F ) are presented. ( H ) Cells cultured with 50mM glucose for 6 days were treated with Calcein AM (1X) and CoCl2 (1x). Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( I ) Quantified data from panel ( H ) are presented. ( J ) TEM was performed to evaluate mitochondrial ultrastructure after 6 days of incubation with 50mM glucose. Scale bar = 2μm. ( K ) Quantified data from panel ( J ) are presented. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

    doi: 10.2147/OPTH.S521660

    Figure Lengend Snippet: TRAP1 rescues mitochondrial damage under high-glucose stimulation. ( A ) The left panel depicts Western blot analysis of TRAP1 overexpression and knockdown cell lines. The right panel shows the quantified data. (n=3) ( B ) Total RNA was extracted from ARPE-19 cells overexpressing or knockdown for TRAP1, followed by qRT-PCR to assess TRAP1 mRNA expression. (n=3) ( C ) Immunofluorescence staining of TRAP1 (red) and Tom20 (green) demonstrates their colocalization in TRAP1 OE and shTRAP1 cells. Cell nuclei were stained with DAPI (blue). Scale bar = 20μm. (n=3) ( D )The upper panel illustrates cell viability of NC group, cells transduced with overexpression empty virus, and cells transduced with knockdown empty virus cultured for 6 days under 50mM glucose and control conditions. The lower panel represents cell viability of NC, TRAP1 OE , and shTRAP1 cells cultured for 6 days under 50mM glucose and control conditions. (n=4) ( E ) Intracellular ROS levels were measured after 6 days of incubation with 50mM glucose. (n=3) ( F ) Cells cultured with 50mM glucose for 6 days were subjected to JC-1 staining to assess Δψ m. Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( G ) Quantified data from panel ( F ) are presented. ( H ) Cells cultured with 50mM glucose for 6 days were treated with Calcein AM (1X) and CoCl2 (1x). Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( I ) Quantified data from panel ( H ) are presented. ( J ) TEM was performed to evaluate mitochondrial ultrastructure after 6 days of incubation with 50mM glucose. Scale bar = 2μm. ( K ) Quantified data from panel ( J ) are presented. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

    Techniques: Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Transduction, Virus, Cell Culture, Control, Incubation, Fluorescence, Microscopy

    Knocking down TRAP1 affects oxidative stress and related mitochondrial functions in ARPE-19 cells. ( A ) Volcano plot of differentially expressed genes (DEGs) between shTRAP1 and NC, with yellow representing significantly upregulated genes and blue representing significantly downregulated genes (|log2 fold change|>1, log10 adjusted p-values<0.05). ( B ) Heatmap of DEGs between shTRAP1 and NC. ( C ) Venn diagram showing a high overlap between DEGs in the shTRAP1 group and oxidative stress-related genes (OS) and diabetic retinopathy-related risk genes (DR). ( D ) Gene Ontology (GO) enrichment analysis of DEGs. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

    doi: 10.2147/OPTH.S521660

    Figure Lengend Snippet: Knocking down TRAP1 affects oxidative stress and related mitochondrial functions in ARPE-19 cells. ( A ) Volcano plot of differentially expressed genes (DEGs) between shTRAP1 and NC, with yellow representing significantly upregulated genes and blue representing significantly downregulated genes (|log2 fold change|>1, log10 adjusted p-values<0.05). ( B ) Heatmap of DEGs between shTRAP1 and NC. ( C ) Venn diagram showing a high overlap between DEGs in the shTRAP1 group and oxidative stress-related genes (OS) and diabetic retinopathy-related risk genes (DR). ( D ) Gene Ontology (GO) enrichment analysis of DEGs. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs.

    Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

    Techniques:

    TRAP1 alleviates high glucose-induced cellular damage by inhibiting mitochondrial ferroptosis.( A ) Activity of shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1, Z-VAD, 3-MA, and Nec-1. (n=3) ( B ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=4) ( C ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( D ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( E ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( F ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. R-BODIPY: Reduced BODIPY(red). O-BODIPY: Oxidized BODIPY(green). Right panel: Quantitative data. Scale bar=20μm. (n=3) ( G ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=4) ( H ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( I ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( J ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( K ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. Right panel: Quantitative data. Scale bar= 20μm. (n=3) ( L ) In Control, OE, and OE+HG groups of ARPE19 cells, immunoprecipitation (IP) was performed using TRAP1 antibody, and the enriched proteins were analyzed by Western Blot with antibodies against ACSL1, ACSL4, and CYB5R1. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

    doi: 10.2147/OPTH.S521660

    Figure Lengend Snippet: TRAP1 alleviates high glucose-induced cellular damage by inhibiting mitochondrial ferroptosis.( A ) Activity of shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1, Z-VAD, 3-MA, and Nec-1. (n=3) ( B ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=4) ( C ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( D ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( E ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( F ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. R-BODIPY: Reduced BODIPY(red). O-BODIPY: Oxidized BODIPY(green). Right panel: Quantitative data. Scale bar=20μm. (n=3) ( G ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=4) ( H ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( I ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( J ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( K ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. Right panel: Quantitative data. Scale bar= 20μm. (n=3) ( L ) In Control, OE, and OE+HG groups of ARPE19 cells, immunoprecipitation (IP) was performed using TRAP1 antibody, and the enriched proteins were analyzed by Western Blot with antibodies against ACSL1, ACSL4, and CYB5R1. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

    Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

    Techniques: Activity Assay, Concentration Assay, Fluorescence, Microscopy, Control, Immunoprecipitation, Western Blot

    Changes in TRAP1, P-ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A-C) TRAP1, P-ERK1/2/ERK1/2, CDC25C and β-actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor-associated protein 1; ERK1/2, extracellular-regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

    Journal: Oncology Letters

    Article Title: Luteolin potentiates low-dose oxaliplatin-induced inhibitory effects on cell proliferation in gastric cancer by inducing G 2 /M cell cycle arrest and apoptosis

    doi: 10.3892/ol.2021.13134

    Figure Lengend Snippet: Changes in TRAP1, P-ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A-C) TRAP1, P-ERK1/2/ERK1/2, CDC25C and β-actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor-associated protein 1; ERK1/2, extracellular-regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

    Article Snippet: The primary antibodies used were: β-actin mouse monoclonal antibody (cat. no. TA-09; 1:2,000; OriGene Technologies, Inc.), Bcl-2 rabbit polyclonal antibody (cat. no. ab196495, 1:1,000; Abcam), BCL-2-associated X protein (Bax) rabbit monoclonal antibody (cat. no. ab182734; 1:1,000; Abcam), cyclin A2 rabbit monoclonal antibody (cat. no. ab181591; 1:2,000; Abcam), cyclin B1 rabbit monoclonal antibody (cat. no. ab32053; 1:1,000; Abcam), cyclin-dependent kinase-1 (CDK1) rabbit monoclonal antibody (cat. no. ab133327; 1:20,000; Abcam), tumor necrosis factor receptor-associated protein 1 (TRAP1) rabbit polyclonal antibody (cat. no. 10325-1-AP; 1:2,000; ProteinTech Group, Inc.), cell division cycle 25 homolog C (CDC25C) mouse monoclonal antibody (cat. no. 66912-1-lg; 1:2,000; ProteinTech Group, Inc.), extracellular-regulated protein kinases1/2 (ERK1/2) rabbit polyclonal antibody (cat. no. 9102s; 1:1,000; Cell Signaling Technology, Inc.) and p-ERK1/2 mouse monoclonal antibody (cat. no. 9106s; 1:1,000; Cell Signaling Technology, Inc.).

    Techniques: Expressing, Western Blot

    Figure 3. Changes in TRAP1, P‑ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A‑C) TRAP1, P‑ERK1/2/ERK1/2, CDC25C and β‑actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor‑associated protein 1; ERK1/2, extracellular‑regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

    Journal: Oncology letters

    Article Title: Luteolin potentiates low-dose oxaliplatin-induced inhibitory effects on cell proliferation in gastric cancer by inducing G 2 /M cell cycle arrest and apoptosis.

    doi: 10.3892/ol.2021.13134

    Figure Lengend Snippet: Figure 3. Changes in TRAP1, P‑ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A‑C) TRAP1, P‑ERK1/2/ERK1/2, CDC25C and β‑actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor‑associated protein 1; ERK1/2, extracellular‑regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

    Article Snippet: The primary antibodies used were: β‐actin mouse monoclonal antibody (cat. no. TA‐09; 1:2,000; OriGene Technologies, Inc.), Bcl‐2 rabbit polyclonal antibody (cat. no. ab196495, 1:1,000; Abcam), BCL‐2‐associated X protein (Bax) rabbit monoclonal antibody (cat. no. ab182734; 1:1,000; Abcam), cyclin A2 rabbit monoclonal antibody (cat. no. ab181591; 1:2,000; Abcam), cyclin B1 rabbit monoclonal antibody (cat. no. ab32053; 1:1,000; Abcam), cyclin‐dependent kinase‐1 (CDK1) rabbit monoclonal antibody (cat. no. ab133327; 1:20,000; Abcam), tumor necrosis factor receptor‐associated protein 1 (TRAP1) rabbit polyclonal anti‐ body (cat. no. 10325‐1‐AP; 1:2,000; ProteinTech Group, Inc.), cell division cycle 25 homolog C (CDC25C) mouse monoclonal antibody (cat. no. 66912‐1‐lg; 1:2,000; ProteinTech Group, Inc.), extracellular‐regulated protein kinases1/2 (ERK1/2) rabbit polyclonal antibody (cat. no. 9102s; 1:1,000; Cell Signaling Technology, Inc.) and p‐ERK1/2 mouse monoclonal antibody (cat. no. 9106s; 1:1,000; Cell Signaling Technology, Inc.).

    Techniques: Expressing, Western Blot

    Changes in TRAP1, P-ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A-C) TRAP1, P-ERK1/2/ERK1/2, CDC25C and β-actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor-associated protein 1; ERK1/2, extracellular-regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

    Journal: Oncology Letters

    Article Title: Luteolin potentiates low-dose oxaliplatin-induced inhibitory effects on cell proliferation in gastric cancer by inducing G 2 /M cell cycle arrest and apoptosis

    doi: 10.3892/ol.2021.13134

    Figure Lengend Snippet: Changes in TRAP1, P-ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A-C) TRAP1, P-ERK1/2/ERK1/2, CDC25C and β-actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor-associated protein 1; ERK1/2, extracellular-regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

    Article Snippet: The primary antibodies used were: β-actin mouse monoclonal antibody (cat. no. TA-09; 1:2,000; OriGene Technologies, Inc.), Bcl-2 rabbit polyclonal antibody (cat. no. ab196495, 1:1,000; Abcam), BCL-2-associated X protein (Bax) rabbit monoclonal antibody (cat. no. ab182734; 1:1,000; Abcam), cyclin A2 rabbit monoclonal antibody (cat. no. ab181591; 1:2,000; Abcam), cyclin B1 rabbit monoclonal antibody (cat. no. ab32053; 1:1,000; Abcam), cyclin-dependent kinase-1 (CDK1) rabbit monoclonal antibody (cat. no. ab133327; 1:20,000; Abcam), tumor necrosis factor receptor-associated protein 1 (TRAP1) rabbit polyclonal antibody (cat. no. 10325-1-AP; 1:2,000; ProteinTech Group, Inc.), cell division cycle 25 homolog C (CDC25C) mouse monoclonal antibody (cat. no. 66912-1-lg; 1:2,000; ProteinTech Group, Inc.), extracellular-regulated protein kinases1/2 (ERK1/2) rabbit polyclonal antibody (cat. no. 9102s; 1:1,000; Cell Signaling Technology, Inc.) and p-ERK1/2 mouse monoclonal antibody (cat. no. 9106s; 1:1,000; Cell Signaling Technology, Inc.).

    Techniques: Expressing, Western Blot

    OMM severing, IMM ubiquitination and distribution of TRAP1 and CPOX in Acn-treated HCT116 and HeLa cells. (A–C) Examples of mitochondria with severed OMM from WT HCT116 (A and B) and WT HeLa (C) cells treated with Acn for 6 h and immunostained to detect conjugated Ub (FK2; green in A–C), Tom20 (blue in A–C), Tom40 (red in A), Fis1 (red in B), and SDHA (red in C). Scale bars represent 1 µm. (D) Quantification of circular mitochondrial ubiquitination in WT HeLa cells treated as indicated in the figure. Data are represented by mean ± SD; n = 3 ( n = 60 cells per experiment). *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal-Wallis with Dunn post-hoc analysis (α = 0.05). (E) Total cell lysates obtained from HeLa (cervical adenocarcinoma), HCT116 (colorectal carcinoma), H4 (neuroglioma), HepG2 (hepatocellular carcinoma), SH-SY5Y (neuroblastoma), U251 (glioblastoma), A549 (lung epithelial carcinoma), and HUH7 (hepatocellular carcinoma) were subjected to Western blot to detect Parkin. The upper “Parkin” panel show blots detected with a standard ECL detection reagent (Pico), and the Parkin panel second from the top was detected using a more sensitive ECL detection reagent (Femto). Rat brain lysate was included as a reference. Tom20 was used as loading control. Numbers in the bottom panel indicate expression of Parkin in all analyzed cells relative to SH-SY5Y neuroblastoma cells that showed the highest expression of this protein. The values were normalized to Tom20 levels. (F–K) Distribution and expression of TRAP1 and CPOX was analyzed in WT and Drp1 −/− HCT116 and WT HeLa cells. WT (F) and Drp1 −/− HCT116 (G and H) cells and WT HeLa cells (I and J) were treated with DMSO (G and I) or Acn (F, H, and J) for 6 h followed by immunostaining to detect cytochrome c (Cyt. c; green), Tom20 (blue), TRAP1 (red in F), and CPOX (red in G and H). Detail images are from areas marked with white rectangles. Scale bars represent 20 µm in F–J; 2 µm in detail images in F, G, and H; and 5 µm in detail images in I and J. Arrowheads in F indicate mitochondria with high cytochrome c/low TRAP1 (red), high TRAP1/low cytochrome c (yellow), and high cytochrome c/high TRAP1 (blue). (K) Expression levels of CPOX, TRAP1, and cytochrome c in WT HeLa cells and WT, ATG5 −/− , and Drp1 −/− HCT116 cells was analyzed by Western blot. Tom20 was used as a loading control. Two exposures of CPOX blot are shown.

    Journal: The Journal of Cell Biology

    Article Title: Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination

    doi: 10.1083/jcb.202006043

    Figure Lengend Snippet: OMM severing, IMM ubiquitination and distribution of TRAP1 and CPOX in Acn-treated HCT116 and HeLa cells. (A–C) Examples of mitochondria with severed OMM from WT HCT116 (A and B) and WT HeLa (C) cells treated with Acn for 6 h and immunostained to detect conjugated Ub (FK2; green in A–C), Tom20 (blue in A–C), Tom40 (red in A), Fis1 (red in B), and SDHA (red in C). Scale bars represent 1 µm. (D) Quantification of circular mitochondrial ubiquitination in WT HeLa cells treated as indicated in the figure. Data are represented by mean ± SD; n = 3 ( n = 60 cells per experiment). *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal-Wallis with Dunn post-hoc analysis (α = 0.05). (E) Total cell lysates obtained from HeLa (cervical adenocarcinoma), HCT116 (colorectal carcinoma), H4 (neuroglioma), HepG2 (hepatocellular carcinoma), SH-SY5Y (neuroblastoma), U251 (glioblastoma), A549 (lung epithelial carcinoma), and HUH7 (hepatocellular carcinoma) were subjected to Western blot to detect Parkin. The upper “Parkin” panel show blots detected with a standard ECL detection reagent (Pico), and the Parkin panel second from the top was detected using a more sensitive ECL detection reagent (Femto). Rat brain lysate was included as a reference. Tom20 was used as loading control. Numbers in the bottom panel indicate expression of Parkin in all analyzed cells relative to SH-SY5Y neuroblastoma cells that showed the highest expression of this protein. The values were normalized to Tom20 levels. (F–K) Distribution and expression of TRAP1 and CPOX was analyzed in WT and Drp1 −/− HCT116 and WT HeLa cells. WT (F) and Drp1 −/− HCT116 (G and H) cells and WT HeLa cells (I and J) were treated with DMSO (G and I) or Acn (F, H, and J) for 6 h followed by immunostaining to detect cytochrome c (Cyt. c; green), Tom20 (blue), TRAP1 (red in F), and CPOX (red in G and H). Detail images are from areas marked with white rectangles. Scale bars represent 20 µm in F–J; 2 µm in detail images in F, G, and H; and 5 µm in detail images in I and J. Arrowheads in F indicate mitochondria with high cytochrome c/low TRAP1 (red), high TRAP1/low cytochrome c (yellow), and high cytochrome c/high TRAP1 (blue). (K) Expression levels of CPOX, TRAP1, and cytochrome c in WT HeLa cells and WT, ATG5 −/− , and Drp1 −/− HCT116 cells was analyzed by Western blot. Tom20 was used as a loading control. Two exposures of CPOX blot are shown.

    Article Snippet: Antibodies used for Western blotting were anti-Tom20 polyclonal antibody (Proteintech, 1:10,000; catalog no. PTG-11802-AP), anti-Tom40 polyclonal antibody (Proteintech, 1:10,000; catalog no. PTG-18409-AP), anti-Ub FK2 mAb (Sigma/Millipore, 1:2,000; catalog no. SIG-04-263), anti-Ub mAb (Santa Cruz Biotechnology; clone P4D1, 1:5,000; catalog no. sc-8017), anti-Ub K48 chain rabbit mAb antibody (Sigma/Millipore; clone Apu2, 1:2,000; catalog no. SIG-05-1307), anti-Ub K63 chain rabbit mAb (Sigma/Millipore; clone Apu3, 1:1,000; catalog no. SIG-05-1308), anti-ATP5a1 polyclonal antibody (Proteintech; 1:5,000; catalog no. PTG-14676-1-AP), anti-ATP6 polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-55313-1-AP), anti-ATP8 polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-26723-1-AP), anti-CPOX polyclonal antibody (Proteintech; 1:5,000; catalog no. PTG-12211-1-AP), anti-TRAP1 polyclonal antibody (Proteintech; 1:10,000; catalog no. PTG-10325-1-AP), anti-human OXPHOS mAb cocktail (Abcam; 1:2,000; catalog no. ab110411), anti-p62/SQTS polyclonal antibody (Proteintech; 1:10,000; catalog no. PTG-18420-1-AP), anti-LC3 polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-14600-1-AP), anti-Opa1 mAb (BD Biosciences; 1:2,000; catalog no. PMG-623606), anti-Mfn2 rabbit mAb (Cell Signaling Technology; clone d2d10; 1:2,500; catalog no. CST-9482), anti-Drp1 mAb (BD Biosciences; 1:2,000; catalog no. PMG-611113), anti-Parkin mAb (Cell Signaling Technology; 1:2,000; catalog no. CST-4211), anti-Bax polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-50599-2-Ig), anti-Bak polyclonal antibody (Sigma-Aldrich; 1:1,000; catalog no. SIG-ABC12), anti-Caspase 1 rabbit mAb (Cell Signaling Technology; 1:2,500; catalog no. CST-2225S), anti-caspase 9 rabbit mAb (Cell Signaling Technology; clone C9; 1:2,000; catalog no. CST-9508), anti-PARP polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-13371-1-AP), anti-β-actin polyclonal antibody (Cell Signaling Technology; 1–10,000; catalog no. CST-4967S), and anti-HA-tag mAb (Invitrogen; 1:5,000; catalog no. 26183).

    Techniques: Ubiquitin Proteomics, Control, Western Blot, Expressing, Immunostaining

    Identification of mitochondrial proteins ubiquitinated in cells with reduced MTF. (A) Summary of mass spectrometry analyzes of ubiquitinated proteins enriched in membrane fractions of Acn-treated versus control (DMSO-treated) cells. For the complete dataset, see . (B–D) Mitochondria-enriched HM and LM fractions from DMSO (control)– or Acn-treated WT (B) or ATG5 −/− HCT116 (C) cells were subjected to Ub pull-down (PD) under denaturing conditions and analyzed by Western blot as indicated in the figure. Inputs (3% of samples used for the Ub pull-down) are shown in D. High-molecular-weight (apparently ubiquitinated) subsets of CPOX, TRAP1, and Mfn2 are marked with a red “Ub.” Upon stress, Mfn2 is degraded by the UPS ( ; ), and ubiquitination of this protein in Acn-treated cells was used as a reference. (E and F) WT HCT116 cells treated with DMSO (control; E) or Acn (F) were labeled to detect cytochrome c (Cyt. c; green), CPOX (red), and Tom20 (blue). Arrowheads in F indicate examples of high–cytochrome c/high-CPOX mitochondria. Detail images are from areas outlined with light green rectangles. Scale bars represent 20 µm (2 µm in detail images). (G) Overlap among cytochrome c, CPOX, and Tom20 in control and Acn-treated WT and Drp1 −/− and Acn plus CP–treated WTHCT116 cells was estimated from maximum intensity projection images (exemplified in E and F). The values are shown as Pearson’s correlation coefficient. Data are represented as mean ± SD; n = 40 cells per condition. **, P < 0.01 versus control in WT HCT116 cells (all other samples were not significant); one-way ANOVA with Bonferroni correction (α = 0.05).

    Journal: The Journal of Cell Biology

    Article Title: Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination

    doi: 10.1083/jcb.202006043

    Figure Lengend Snippet: Identification of mitochondrial proteins ubiquitinated in cells with reduced MTF. (A) Summary of mass spectrometry analyzes of ubiquitinated proteins enriched in membrane fractions of Acn-treated versus control (DMSO-treated) cells. For the complete dataset, see . (B–D) Mitochondria-enriched HM and LM fractions from DMSO (control)– or Acn-treated WT (B) or ATG5 −/− HCT116 (C) cells were subjected to Ub pull-down (PD) under denaturing conditions and analyzed by Western blot as indicated in the figure. Inputs (3% of samples used for the Ub pull-down) are shown in D. High-molecular-weight (apparently ubiquitinated) subsets of CPOX, TRAP1, and Mfn2 are marked with a red “Ub.” Upon stress, Mfn2 is degraded by the UPS ( ; ), and ubiquitination of this protein in Acn-treated cells was used as a reference. (E and F) WT HCT116 cells treated with DMSO (control; E) or Acn (F) were labeled to detect cytochrome c (Cyt. c; green), CPOX (red), and Tom20 (blue). Arrowheads in F indicate examples of high–cytochrome c/high-CPOX mitochondria. Detail images are from areas outlined with light green rectangles. Scale bars represent 20 µm (2 µm in detail images). (G) Overlap among cytochrome c, CPOX, and Tom20 in control and Acn-treated WT and Drp1 −/− and Acn plus CP–treated WTHCT116 cells was estimated from maximum intensity projection images (exemplified in E and F). The values are shown as Pearson’s correlation coefficient. Data are represented as mean ± SD; n = 40 cells per condition. **, P < 0.01 versus control in WT HCT116 cells (all other samples were not significant); one-way ANOVA with Bonferroni correction (α = 0.05).

    Article Snippet: Antibodies used for Western blotting were anti-Tom20 polyclonal antibody (Proteintech, 1:10,000; catalog no. PTG-11802-AP), anti-Tom40 polyclonal antibody (Proteintech, 1:10,000; catalog no. PTG-18409-AP), anti-Ub FK2 mAb (Sigma/Millipore, 1:2,000; catalog no. SIG-04-263), anti-Ub mAb (Santa Cruz Biotechnology; clone P4D1, 1:5,000; catalog no. sc-8017), anti-Ub K48 chain rabbit mAb antibody (Sigma/Millipore; clone Apu2, 1:2,000; catalog no. SIG-05-1307), anti-Ub K63 chain rabbit mAb (Sigma/Millipore; clone Apu3, 1:1,000; catalog no. SIG-05-1308), anti-ATP5a1 polyclonal antibody (Proteintech; 1:5,000; catalog no. PTG-14676-1-AP), anti-ATP6 polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-55313-1-AP), anti-ATP8 polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-26723-1-AP), anti-CPOX polyclonal antibody (Proteintech; 1:5,000; catalog no. PTG-12211-1-AP), anti-TRAP1 polyclonal antibody (Proteintech; 1:10,000; catalog no. PTG-10325-1-AP), anti-human OXPHOS mAb cocktail (Abcam; 1:2,000; catalog no. ab110411), anti-p62/SQTS polyclonal antibody (Proteintech; 1:10,000; catalog no. PTG-18420-1-AP), anti-LC3 polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-14600-1-AP), anti-Opa1 mAb (BD Biosciences; 1:2,000; catalog no. PMG-623606), anti-Mfn2 rabbit mAb (Cell Signaling Technology; clone d2d10; 1:2,500; catalog no. CST-9482), anti-Drp1 mAb (BD Biosciences; 1:2,000; catalog no. PMG-611113), anti-Parkin mAb (Cell Signaling Technology; 1:2,000; catalog no. CST-4211), anti-Bax polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-50599-2-Ig), anti-Bak polyclonal antibody (Sigma-Aldrich; 1:1,000; catalog no. SIG-ABC12), anti-Caspase 1 rabbit mAb (Cell Signaling Technology; 1:2,500; catalog no. CST-2225S), anti-caspase 9 rabbit mAb (Cell Signaling Technology; clone C9; 1:2,000; catalog no. CST-9508), anti-PARP polyclonal antibody (Proteintech; 1:2,000; catalog no. PTG-13371-1-AP), anti-β-actin polyclonal antibody (Cell Signaling Technology; 1–10,000; catalog no. CST-4967S), and anti-HA-tag mAb (Invitrogen; 1:5,000; catalog no. 26183).

    Techniques: Mass Spectrometry, Membrane, Control, Western Blot, High Molecular Weight, Ubiquitin Proteomics, Labeling