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Copper deprivation induced by SLC31A11 knockdown triggers the upregulation of SLC7A11. ( A ) Confocal imaging of Cu-probe and Mito-tracker in the NC and <t>SLC31A1</t> knockdown AsPC-1 cells. Hoechst (blue) is used as a nuclear counterstain. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 2 μm. ( B ) Total Fe level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( C ) Volcano plot of gene expression (the SLC31A1 knockdown [shRNA#2] versus the control; log2(fold change) ≥1; p < 0.05 between SLC31A1 knockdown and NC AsPC-1 cells. ( D ) GO analysis of differentially expressed genes between SLC31A1 knockdown (shRNA#2) and NC AsPC-1 cells. ( E-G ) Western blot analysis of the indicated protein levels in the NC and SLC31A1 knockdown AsPC-1 (E), MiaPaCa-2 (F), and CFPAC-1 (G) cells. ( H and I ) GPX4 (H) or FSP-1 activity (I) was tested in the NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( J ) qPCR analysis of SLC31A1 and SLC7A11 gene expression in NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( K ) qPCR analysis of SLC31A1 and SLC7A11 gene expression in NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( L ) GSH level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( M ) Cystine uptake level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Slc31a1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 27 article reviews

slc31a1 - by Bioz Stars, 2026-09

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1) Product Images from "Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11"

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

Journal: Redox Biology

doi: 10.1016/j.redox.2026.104130

Figure Legend Snippet: Copper deprivation induced by SLC31A11 knockdown triggers the upregulation of SLC7A11. ( A ) Confocal imaging of Cu-probe and Mito-tracker in the NC and SLC31A1 knockdown AsPC-1 cells. Hoechst (blue) is used as a nuclear counterstain. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 2 μm. ( B ) Total Fe level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( C ) Volcano plot of gene expression (the SLC31A1 knockdown [shRNA#2] versus the control; log2(fold change) ≥1; p < 0.05 between SLC31A1 knockdown and NC AsPC-1 cells. ( D ) GO analysis of differentially expressed genes between SLC31A1 knockdown (shRNA#2) and NC AsPC-1 cells. ( E-G ) Western blot analysis of the indicated protein levels in the NC and SLC31A1 knockdown AsPC-1 (E), MiaPaCa-2 (F), and CFPAC-1 (G) cells. ( H and I ) GPX4 (H) or FSP-1 activity (I) was tested in the NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( J ) qPCR analysis of SLC31A1 and SLC7A11 gene expression in NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( K ) qPCR analysis of SLC31A1 and SLC7A11 gene expression in NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( L ) GSH level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( M ) Cystine uptake level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Techniques Used: Knockdown, Imaging, Gene Expression, shRNA, Control, Western Blot, Activity Assay

Copper deprivation induced by SLC31A1 knockdown attenuates ferroptosis. ( A ) Cell viability of AsPC-1 cells treated with different doses of RSL3, ML162, ML210, or erastin in NC and SLC31A1 knockdown AsPC-1 cells. ( B ) Cell viability of AsPC-1 cells treated with different doses of CDDP, staurosporine, paclitaxel, bortezomib, JTC-801, or elesclomol-Cu in NC and SLC31A1 knockdown AsPC-1 cells. ( C-E ) Propidium iodide (PI) staining of NC and SLC31A1 knockdown AsPC-1 (C), MiaPaCa-2 (D), and CFPAC-1 (E) cells treated with RSL3 at the indicated concentrations in the presence or absence of ferroptosis inhibitors (ferrostatin-1/Fer-1, 5 μM or desferrioxamine/DFO, 20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( F ) Lipid peroxidation of NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. (G) Transmission electron microscopy of NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 (2.5 μM). Scale bar: 2 μm or 500 nm. ( H ) Confocal imaging of Cu-probe and Mito-tracker in the NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1. Hoechst (blue) is used as a nuclear counterstain. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 2 μm. ( I ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1. ( J ) Cell viability of NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1, followed by treatment with different doses of RSL3 or erastin. ( K ) PI staining of NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1, followed by treatment with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( L ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or SLC31A1 WT or SLC31A1 M154A plasmid. ( M ) PI staining of SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or SLC31A1 WT or SLC31A1 M154A plasmid, followed by treatment with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test.

Figure Legend Snippet: Copper deprivation induced by SLC31A1 knockdown attenuates ferroptosis. ( A ) Cell viability of AsPC-1 cells treated with different doses of RSL3, ML162, ML210, or erastin in NC and SLC31A1 knockdown AsPC-1 cells. ( B ) Cell viability of AsPC-1 cells treated with different doses of CDDP, staurosporine, paclitaxel, bortezomib, JTC-801, or elesclomol-Cu in NC and SLC31A1 knockdown AsPC-1 cells. ( C-E ) Propidium iodide (PI) staining of NC and SLC31A1 knockdown AsPC-1 (C), MiaPaCa-2 (D), and CFPAC-1 (E) cells treated with RSL3 at the indicated concentrations in the presence or absence of ferroptosis inhibitors (ferrostatin-1/Fer-1, 5 μM or desferrioxamine/DFO, 20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( F ) Lipid peroxidation of NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. (G) Transmission electron microscopy of NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 (2.5 μM). Scale bar: 2 μm or 500 nm. ( H ) Confocal imaging of Cu-probe and Mito-tracker in the NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1. Hoechst (blue) is used as a nuclear counterstain. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 2 μm. ( I ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1. ( J ) Cell viability of NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1, followed by treatment with different doses of RSL3 or erastin. ( K ) PI staining of NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1, followed by treatment with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( L ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or SLC31A1 WT or SLC31A1 M154A plasmid. ( M ) PI staining of SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or SLC31A1 WT or SLC31A1 M154A plasmid, followed by treatment with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test.

Techniques Used: Knockdown, Staining, Transmission Assay, Electron Microscopy, Imaging, shRNA, Transfection, Plasmid Preparation, Western Blot

SLC7A11 is essential for copper deprivation-mediated ferroptosis resistance. ( A ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC), SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells. ( B ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells treated with or without TM (50 μM) for 24 h. ( C ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Propidium iodide (PI) staining of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( D ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). PI staining of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( E ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Lipid peroxidation of the AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( F ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( G ) PI staining of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of BSO (500 μM) with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( H ) PI staining images of the indicated AsPC-1 cells treated with or without BSO (500 μM) plus TM sulfate (50 μM) with RSL3 at the indicated concentrations with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test.

Figure Legend Snippet: SLC7A11 is essential for copper deprivation-mediated ferroptosis resistance. ( A ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC), SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells. ( B ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells treated with or without TM (50 μM) for 24 h. ( C ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Propidium iodide (PI) staining of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( D ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). PI staining of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( E ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Lipid peroxidation of the AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( F ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( G ) PI staining of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of BSO (500 μM) with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( H ) PI staining images of the indicated AsPC-1 cells treated with or without BSO (500 μM) plus TM sulfate (50 μM) with RSL3 at the indicated concentrations with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test.

Techniques Used: Knockdown, shRNA, Transfection, Western Blot, Staining

SLC7A11 is essential for copper deprivation-mediated ferroptosis resistance. ( A ) Vector or flag-SLC7A11 overexpressed AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells. ( B ) AsPC-1 cells were transfected with a vector or a plasmid encoding Flag-SLC7A11 and then treated with TM (50 μM) for 24h. Western blot analysis of lysates from the indicated AsPC-1 cells. ( C ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11) with or without transfection with a blank vector or a vector encoding SLC7A11. PI staining of indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of Fer-1 (2.5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( D ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11) with or without transfection with a blank vector or a vector encoding SLC7A11. PI staining images of indicated AsPC-1 cells treated with RSL3 (0.25 μM) or TM (50 μM) in the presence or absence of Fer-1 (2.5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Figure Legend Snippet: SLC7A11 is essential for copper deprivation-mediated ferroptosis resistance. ( A ) Vector or flag-SLC7A11 overexpressed AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells. ( B ) AsPC-1 cells were transfected with a vector or a plasmid encoding Flag-SLC7A11 and then treated with TM (50 μM) for 24h. Western blot analysis of lysates from the indicated AsPC-1 cells. ( C ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11) with or without transfection with a blank vector or a vector encoding SLC7A11. PI staining of indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of Fer-1 (2.5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( D ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11) with or without transfection with a blank vector or a vector encoding SLC7A11. PI staining images of indicated AsPC-1 cells treated with RSL3 (0.25 μM) or TM (50 μM) in the presence or absence of Fer-1 (2.5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Techniques Used: Plasmid Preparation, Transfection, Western Blot, Knockdown, shRNA, Staining

Copper deprivation upregulates SLC7A11 and activates AMPK-NRF2 axis. ( A ) Volcano plot of NRF2 target genes (shSLC31A1 VS shNC) generated using the data from C. ( B , C ) ChIP analyses of NRF2 binding in the SLC7A11 promoter in NRF2 knockdown AsPC-1 cells (B) or SLC31A1 knockdown AsPC-1 cells (C). ( D-G ) The oxygen consumption rate (OCR) in NC and SLC31A1 knockdown AsPC-1 cells by Seahorse assay (n = 8/group). Quantification of basal respiration (E) and ATP-linked respiration (F), the uncoupler FCCP (maximal) or the electron transport inhibitor antimycin A-baseline (G) in NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 8. Statistical significance was determined using a one-way ANOVA test. ( H ) COX activity level in the NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( I–K ) ATP level in the NC and SLC31A1 knockdown AsPC-1(I), MiaPaCa-2 (J), and CFPAC-1 (K) cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( L ) ATP level in the AsPC-1 cells treated with TM (50 μM) or TEPA (1 mM) for 24 h. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( M ) AMP/ATP ratio detected by HPLC in NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using an unpaired T test. ( N ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells. ( O ) Western blot analysis of lysates from AsPC-1 cells treated with TM at the indicated concentrations for 24 h. ( P ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with blank vector or vector encoding SLC31A1. ( Q ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with blank vector or of SLC31A1 WT- or SLC31A1 M154A.

Figure Legend Snippet: Copper deprivation upregulates SLC7A11 and activates AMPK-NRF2 axis. ( A ) Volcano plot of NRF2 target genes (shSLC31A1 VS shNC) generated using the data from C. ( B , C ) ChIP analyses of NRF2 binding in the SLC7A11 promoter in NRF2 knockdown AsPC-1 cells (B) or SLC31A1 knockdown AsPC-1 cells (C). ( D-G ) The oxygen consumption rate (OCR) in NC and SLC31A1 knockdown AsPC-1 cells by Seahorse assay (n = 8/group). Quantification of basal respiration (E) and ATP-linked respiration (F), the uncoupler FCCP (maximal) or the electron transport inhibitor antimycin A-baseline (G) in NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 8. Statistical significance was determined using a one-way ANOVA test. ( H ) COX activity level in the NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( I–K ) ATP level in the NC and SLC31A1 knockdown AsPC-1(I), MiaPaCa-2 (J), and CFPAC-1 (K) cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( L ) ATP level in the AsPC-1 cells treated with TM (50 μM) or TEPA (1 mM) for 24 h. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( M ) AMP/ATP ratio detected by HPLC in NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using an unpaired T test. ( N ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells. ( O ) Western blot analysis of lysates from AsPC-1 cells treated with TM at the indicated concentrations for 24 h. ( P ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with blank vector or vector encoding SLC31A1. ( Q ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with blank vector or of SLC31A1 WT- or SLC31A1 M154A.

Techniques Used: Generated, Binding Assay, Knockdown, Activity Assay, Western Blot, shRNA, Transfection, Plasmid Preparation

Copper deprivation upregulates SLC7A11 through AMPK-NRF2 axis. ( A ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). ( B ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNFE2L2#2). * indicates a non-specific band. ( C ) SLC7A11 mRNA level in NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2#2). ( D ) Western blot analysis of lysates from AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( E ) SLC7A11 mRNA level in AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( F ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells treated with or without ML385 (5 μM) for 24 h. ( G ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. ( H ) SLC7A11 mRNA level in SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Figure Legend Snippet: Copper deprivation upregulates SLC7A11 through AMPK-NRF2 axis. ( A ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). ( B ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNFE2L2#2). * indicates a non-specific band. ( C ) SLC7A11 mRNA level in NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2#2). ( D ) Western blot analysis of lysates from AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( E ) SLC7A11 mRNA level in AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( F ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells treated with or without ML385 (5 μM) for 24 h. ( G ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. ( H ) SLC7A11 mRNA level in SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Techniques Used: Western Blot, Knockdown, shRNA, Transfection, Plasmid Preparation

Copper deprivation inhibits ferroptosis by the activation of AMPK. ( A ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). Propidium iodide (PI) staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( B ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( C ) PI staining of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of Compound C (10 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Figure Legend Snippet: Copper deprivation inhibits ferroptosis by the activation of AMPK. ( A ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). Propidium iodide (PI) staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( B ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( C ) PI staining of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of Compound C (10 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Techniques Used: Activation Assay, Knockdown, shRNA, Transfection, Staining

Copper deprivation inhibits ferroptosis by the activation of AMPK-NRF2-SLC7A11 pathway. ( A ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( B ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( C ) SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( D ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( E ) PI staining images of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of ML385 (5 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( F ) PI staining of SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid, followed by treatment with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Figure Legend Snippet: Copper deprivation inhibits ferroptosis by the activation of AMPK-NRF2-SLC7A11 pathway. ( A ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( B ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( C ) SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( D ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( E ) PI staining images of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of ML385 (5 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( F ) PI staining of SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid, followed by treatment with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Techniques Used: Activation Assay, Knockdown, Transfection, shRNA, Staining, Plasmid Preparation

SLC31A1 knockdown inhibits ferroptosis in vivo . ( A-D ) Subcutaneous growth of NC and SLC31A1 knockdown AsPC-1 xenografts in nude mice (n = 8) treated with RSL3 (5 mg/kg, i.p., once every other day). ( A ) Tumor growth curves of NC and SLC31A1 knockdown AsPC-1 xenografts in nude mice treated with the indicated agents. Representative images of tumors and tumor weight were shown ( B ). Data are presented as mean ± SD, n = 8 mice per group; Statistical significance was determined using a two-way ANOVA test. ( C ) Total copper content in the tumor sections. Statistical significance was determined using a two-way ANOVA test. ( D and E ) Immunohistochemical staining and relative staining intensity of the indicated proteins in the tumor sections. CC3, cleaved caspase 3. Scale bar: 20 μm. Statistical significance was determined using a two-way ANOVA test.

Figure Legend Snippet: SLC31A1 knockdown inhibits ferroptosis in vivo . ( A-D ) Subcutaneous growth of NC and SLC31A1 knockdown AsPC-1 xenografts in nude mice (n = 8) treated with RSL3 (5 mg/kg, i.p., once every other day). ( A ) Tumor growth curves of NC and SLC31A1 knockdown AsPC-1 xenografts in nude mice treated with the indicated agents. Representative images of tumors and tumor weight were shown ( B ). Data are presented as mean ± SD, n = 8 mice per group; Statistical significance was determined using a two-way ANOVA test. ( C ) Total copper content in the tumor sections. Statistical significance was determined using a two-way ANOVA test. ( D and E ) Immunohistochemical staining and relative staining intensity of the indicated proteins in the tumor sections. CC3, cleaved caspase 3. Scale bar: 20 μm. Statistical significance was determined using a two-way ANOVA test.

Techniques Used: Knockdown, In Vivo, Immunohistochemical staining, Staining

Western Blot:

Article Title: Copper exposure induces autophagy and angiogenesis in liver cell via ATF4 activation independent of TFEB.
Article Snippet: Copper (Cu) pollution has become as a significant global environmental concern, leading to excessive Cu accumulation in humans and disrupting cellular homeostasis.. As a key catalytic cofactor, Cu is involved in essential biological processes, including oxidative stress detoxification, angiogenesis, pigmentation, and peptide hormone production.. However, excessive copper exposure has been linked to various cellular pathological processes.

Immunofluorescence:

Labeling:

Immunoprecipitation:

Article Title: Radiotherapy promotes cuproptosis and synergizes with cuproptosis inducers to overcome tumor radioresistance.
Article Snippet: To identify the pathways enriched in the overlapped upregulated DE genes, we performed pathway overrepresentation analysis against the GO datasets (biological process (BP), cellular component (CC), and molecular function (MF)) using the enrichR package.26 Western blot and oligomerization analysis Cell pellets were lysed using RIPA lysis buffer containing complete mini protease inhibitors (Roche).44 Protein concentrations were measured by a Bicinchoninic Acid Protein Assay (Thermo Scientific) using a FLUOstar Omega microplate reader (BMG Labtech). .. Immunoprecipitation andWestern blot analysis were conducted as previously described.45,46 The primary antibodies and concentrations used in the current study included the following: MT1E (1:1,000, MA5-43655, Fisher), MT1X (1:1,000, 17172-1-AP, Proteintech), BACH1 (1:1,000, 14018-1-AP, Proteintech), NRF2 (1:1,000, 12721S, Cell Signaling), FDX1 (1:1,000, 12592-1-AP, Proteintech), LIAS (1:1,000, 11577-1-AP, Proteintech), Lipoic acid (1:1,000, 437695, Millipore), DLAT (1:1,000, 12362s, Cell Signaling), CTR1 (1:1,000, 13086, Cell Signaling), DMT1 (1:1,000, 20507-1-AP, Proteintech), ETS1 (1:2000, 14069, Cell Signaling), Vinculin (1:10000; V4505, Sigma), b-actin (1:5,000, 3700, Cell Signaling), Bcl-2 (1:1,000, 15071, Cell Signaling), SLC25A39 (1:1,000, 14963-1-AP, Proteintech), and Cleaved Caspase-3 (Asp175) (1:1000, 9661s, Cell Signaling). ..

Incubation:

Article Title: Steroidogenic factor 1 promotes cuproptosis in renal tubular epithelial cells during acute kidney injury
Article Snippet: .. Primary antibodies, namely CTR1 (1:1000, catalog no. 13086S, Cell Signaling Technology, Danvers, MA, USA), FDX1 (1:1000, catalog no. NBP1-89227, Novus, Colorado Springs, CO, USA), and β-actin (1:2000, catalog no. T40104S, Abbart, Shanghai, China) were added, and incubation was conducted overnight in a shaker at 4°C. .. Next, horseradish peroxidase-conjugated anti-mouse IgG (1:3000, catalog no. 7076S, Cell Signaling Technology) and horseradish peroxidase-linked goat anti-rabbit (1:30000, Cat# 7074, RRID: AB_2099233, Cell Signaling Technology) were added.

Membrane:

Article Title: Copper Chaperone Atox1 Protected the Cochlea From Cisplatin by Regulating the Copper Transport Family and Cell Cycle.
Article Snippet: Antioxidant 1 copper chaperone (Atox1) may contribute to preventing DDP cochlear damage by regulating copper transport family and cell cycle proteins.. A rat model of cochlear damage was developed by placing gelatin sponges treated with DDP in the cochlea.. HEI-OC1 cells were treated with 133 μM DDP as a cell model. DDP-induced ototoxicity in rats was confirmed by immunofluorescence (IF) imaging.

Image Search Results

Effects of ovariectomy or estradiol on SIRT5 expression and cuproptosis markers. (A) Micro-CT analysis results of the mice’s distal femur (scale bar: 1 mm). (B) Quantitative results of micro-CT analysis show the bone mineral density (BMD), the ratio of the bone volume to the total volume (BV/TV), the number of trabeculae (Tb.N), and the trabecular thickness (Tb.Th) ( n = 5). (C, D) Serum and bone tissue levels of Cu 2+ in mice ( n = 3). (E, F) Quantitative PCR and Western blotting assay results show the expression of FDX1, SIRT5, CTR1, and ATP7A. (G, H) Representative ICC images show the expression of FDX1 and SIRT5 in mice femur (scale bar = 50 μm for 1G or 25 μm for 1H). (I – L) Quantitative PCR assay results show the mRNA expression levels of Atp7a , Fdx1 , Sirt5 , and Ctr1 in C3H10T1/2 cells. (M) Western blotting assay results show the protein levels of FDX1, SIRT5, CTR1, and ATP7A in C3H10T1/2 cells treated with estradiol. (N) Quantitative results of Western blotting assay show the protein levels of SIRT5 in C3H10T1/2 cells treated with estradiol. (O) Western blotting assay results show the protein levels of SIRT5 in C3H10T1/2 cells treated with tamoxifen. (P) Quantitative results of Western blotting assay show the protein levels of SIRT5 in C3H10T1/2 cells treated with tamoxifen. ns P > 0.05, ∗P < 0.05, and ∗∗P < 0.01.

Journal: Genes & Diseases

Article Title: SIRT5 mediates the pro-osteogenic effects of estrogen through FDX1 demalonylation and cuproptosis inhibition in mesenchymal stem cells

doi: 10.1016/j.gendis.2026.102145

Figure Lengend Snippet: Effects of ovariectomy or estradiol on SIRT5 expression and cuproptosis markers. (A) Micro-CT analysis results of the mice’s distal femur (scale bar: 1 mm). (B) Quantitative results of micro-CT analysis show the bone mineral density (BMD), the ratio of the bone volume to the total volume (BV/TV), the number of trabeculae (Tb.N), and the trabecular thickness (Tb.Th) ( n = 5). (C, D) Serum and bone tissue levels of Cu 2+ in mice ( n = 3). (E, F) Quantitative PCR and Western blotting assay results show the expression of FDX1, SIRT5, CTR1, and ATP7A. (G, H) Representative ICC images show the expression of FDX1 and SIRT5 in mice femur (scale bar = 50 μm for 1G or 25 μm for 1H). (I – L) Quantitative PCR assay results show the mRNA expression levels of Atp7a , Fdx1 , Sirt5 , and Ctr1 in C3H10T1/2 cells. (M) Western blotting assay results show the protein levels of FDX1, SIRT5, CTR1, and ATP7A in C3H10T1/2 cells treated with estradiol. (N) Quantitative results of Western blotting assay show the protein levels of SIRT5 in C3H10T1/2 cells treated with estradiol. (O) Western blotting assay results show the protein levels of SIRT5 in C3H10T1/2 cells treated with tamoxifen. (P) Quantitative results of Western blotting assay show the protein levels of SIRT5 in C3H10T1/2 cells treated with tamoxifen. ns P > 0.05, ∗P < 0.05, and ∗∗P < 0.01.

Article Snippet: Primary antibody against β-actin (AC026), RUNX2 (A2581), and FDX1 (A20895) were ordered from ABclonal (Wuhan, China); SIRT5 (5122-1-AP) and Ubiquitin (10201-2-AP) were ordered from Proteintech (Wuhan, China); Malonyllysine (PTM-901) was ordered from PTM Biolabs (Hongzhou, China); acetyl Lysine (ab80178) was ordered from Abcam (Shanghai, China); ATP7A (TD8506) and CTR1 (T510261F) were ordered from Abmart (Shanghai, China); OPN (sc-10593) was ordered from Santa Cruz Biotechnology (Shanghai, China).

Techniques: Expressing, Micro-CT, Real-time Polymerase Chain Reaction, Western Blot

Effects of copper or SIRT5 activator on osteoporosis in ovariectomized mice. (A) Micro-CT assay results of the distal femur of mice (scale bar = 1 mm). (B) Quantitative results of micro-CT assay show the bone mineral density (BMD), the ratio of the bone volume to the total volume (BV/TV), the number of trabeculae (Tb.N), and the trabecular thickness (Tb.Th) of femoral trabecular bone mass ( n = 5). (C) Representative images of hematoxylin-eosin and Masson’s trichrome staining show the effects of copper, tetrathiomolybdate (TTM, a copper chelator), and/or MC3138 (a SIRT5 activator) on ovariectomy (OVX)-induced bone loss (scale bar = 50 μm). (D, E) Representative images of immunofluorescent staining and quantitative results show the effect of copper and/or TTM on FDX1 affected by OVX. (F, G) Western blotting assay and quantitative results show the effects of copper or TTM on the protein level of FDX1, SIRT5, CTR1, and ATP7A affected by OVX. ns P > 0.05, ∗P < 0.05, and ∗∗P < 0.01.

Journal: Genes & Diseases

Article Title: SIRT5 mediates the pro-osteogenic effects of estrogen through FDX1 demalonylation and cuproptosis inhibition in mesenchymal stem cells

doi: 10.1016/j.gendis.2026.102145

Figure Lengend Snippet: Effects of copper or SIRT5 activator on osteoporosis in ovariectomized mice. (A) Micro-CT assay results of the distal femur of mice (scale bar = 1 mm). (B) Quantitative results of micro-CT assay show the bone mineral density (BMD), the ratio of the bone volume to the total volume (BV/TV), the number of trabeculae (Tb.N), and the trabecular thickness (Tb.Th) of femoral trabecular bone mass ( n = 5). (C) Representative images of hematoxylin-eosin and Masson’s trichrome staining show the effects of copper, tetrathiomolybdate (TTM, a copper chelator), and/or MC3138 (a SIRT5 activator) on ovariectomy (OVX)-induced bone loss (scale bar = 50 μm). (D, E) Representative images of immunofluorescent staining and quantitative results show the effect of copper and/or TTM on FDX1 affected by OVX. (F, G) Western blotting assay and quantitative results show the effects of copper or TTM on the protein level of FDX1, SIRT5, CTR1, and ATP7A affected by OVX. ns P > 0.05, ∗P < 0.05, and ∗∗P < 0.01.

Techniques: Micro-CT, Staining, Western Blot

Effects of SIRT5 on the reduced osteogenic potential and cuproptosis induced by elesclomol (ES, 15 nM) in C3H10T1/2 cells. (A) Western blotting assay results show the levels of RUNX2 in cells treated with ES and/or AdSirt5. (B, C) ALP staining and quantitative results show the ALP activities in cells treated with ES and/or AdSirt5. (D) Western blotting assay results show the level of OPN in cells treated with ES and/or AdSirt5. (E, F) Alizarin red S staining and quantitative results show the mineralization levels in cells treated with ES and/or AdSirt5. (G) Representative transmission electron microscope images show the effect of ES and/or SIRT5 on mitochondria in C3H10T1/2 cells (scale bar = 500 nm). The red arrows indicate the damaged mitochondria, and the white arrows indicate the normal mitochondria. (H) Representative immunofluorescent staining images show the effect of ES and/or SIRT5 on mitochondria in C3H10T1/2 cells (scale bar = 10 μm). (I, J) JC-1 immunofluorescent assay and quantitative results show the effect of ES and/or SIRT5 on mitochondria in C3H10T1/2 cells (scale bar = 50 μm). (K) Copper content assay results show the effect of ES and/or SIRT5 on the medium or cellular level of Cu 2+ . (L, M) Western blotting assay and quantitative results show the effect of ES and/or SIRT5 on the level of FDX1, ATP7A, CTR1, and SIRT5 in C3H10T1/2 cells. ns P > 0.05, ∗P < 0.05, and ∗∗P < 0.01.

Journal: Genes & Diseases

Article Title: SIRT5 mediates the pro-osteogenic effects of estrogen through FDX1 demalonylation and cuproptosis inhibition in mesenchymal stem cells

doi: 10.1016/j.gendis.2026.102145

Figure Lengend Snippet: Effects of SIRT5 on the reduced osteogenic potential and cuproptosis induced by elesclomol (ES, 15 nM) in C3H10T1/2 cells. (A) Western blotting assay results show the levels of RUNX2 in cells treated with ES and/or AdSirt5. (B, C) ALP staining and quantitative results show the ALP activities in cells treated with ES and/or AdSirt5. (D) Western blotting assay results show the level of OPN in cells treated with ES and/or AdSirt5. (E, F) Alizarin red S staining and quantitative results show the mineralization levels in cells treated with ES and/or AdSirt5. (G) Representative transmission electron microscope images show the effect of ES and/or SIRT5 on mitochondria in C3H10T1/2 cells (scale bar = 500 nm). The red arrows indicate the damaged mitochondria, and the white arrows indicate the normal mitochondria. (H) Representative immunofluorescent staining images show the effect of ES and/or SIRT5 on mitochondria in C3H10T1/2 cells (scale bar = 10 μm). (I, J) JC-1 immunofluorescent assay and quantitative results show the effect of ES and/or SIRT5 on mitochondria in C3H10T1/2 cells (scale bar = 50 μm). (K) Copper content assay results show the effect of ES and/or SIRT5 on the medium or cellular level of Cu 2+ . (L, M) Western blotting assay and quantitative results show the effect of ES and/or SIRT5 on the level of FDX1, ATP7A, CTR1, and SIRT5 in C3H10T1/2 cells. ns P > 0.05, ∗P < 0.05, and ∗∗P < 0.01.

Techniques: Western Blot, Staining, Transmission Assay, Microscopy

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation induced by SLC31A11 knockdown triggers the upregulation of SLC7A11. ( A ) Confocal imaging of Cu-probe and Mito-tracker in the NC and SLC31A1 knockdown AsPC-1 cells. Hoechst (blue) is used as a nuclear counterstain. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 2 μm. ( B ) Total Fe level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( C ) Volcano plot of gene expression (the SLC31A1 knockdown [shRNA#2] versus the control; log2(fold change) ≥1; p < 0.05 between SLC31A1 knockdown and NC AsPC-1 cells. ( D ) GO analysis of differentially expressed genes between SLC31A1 knockdown (shRNA#2) and NC AsPC-1 cells. ( E-G ) Western blot analysis of the indicated protein levels in the NC and SLC31A1 knockdown AsPC-1 (E), MiaPaCa-2 (F), and CFPAC-1 (G) cells. ( H and I ) GPX4 (H) or FSP-1 activity (I) was tested in the NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( J ) qPCR analysis of SLC31A1 and SLC7A11 gene expression in NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( K ) qPCR analysis of SLC31A1 and SLC7A11 gene expression in NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( L ) GSH level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( M ) Cystine uptake level in the NC and SLC31A1 knockdown AsPC-1, MiaPaCa-2, and CFPAC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Knockdown, Imaging, Gene Expression, shRNA, Control, Western Blot, Activity Assay

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation induced by SLC31A1 knockdown attenuates ferroptosis. ( A ) Cell viability of AsPC-1 cells treated with different doses of RSL3, ML162, ML210, or erastin in NC and SLC31A1 knockdown AsPC-1 cells. ( B ) Cell viability of AsPC-1 cells treated with different doses of CDDP, staurosporine, paclitaxel, bortezomib, JTC-801, or elesclomol-Cu in NC and SLC31A1 knockdown AsPC-1 cells. ( C-E ) Propidium iodide (PI) staining of NC and SLC31A1 knockdown AsPC-1 (C), MiaPaCa-2 (D), and CFPAC-1 (E) cells treated with RSL3 at the indicated concentrations in the presence or absence of ferroptosis inhibitors (ferrostatin-1/Fer-1, 5 μM or desferrioxamine/DFO, 20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( F ) Lipid peroxidation of NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. (G) Transmission electron microscopy of NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 (2.5 μM). Scale bar: 2 μm or 500 nm. ( H ) Confocal imaging of Cu-probe and Mito-tracker in the NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1. Hoechst (blue) is used as a nuclear counterstain. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 2 μm. ( I ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1. ( J ) Cell viability of NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1, followed by treatment with different doses of RSL3 or erastin. ( K ) PI staining of NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or a vector encoding SLC31A1, followed by treatment with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( L ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or SLC31A1 WT or SLC31A1 M154A plasmid. ( M ) PI staining of SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or SLC31A1 WT or SLC31A1 M154A plasmid, followed by treatment with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Knockdown, Staining, Transmission Assay, Electron Microscopy, Imaging, shRNA, Transfection, Plasmid Preparation, Western Blot

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: SLC7A11 is essential for copper deprivation-mediated ferroptosis resistance. ( A ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC), SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells. ( B ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells treated with or without TM (50 μM) for 24 h. ( C ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Propidium iodide (PI) staining of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( D ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). PI staining of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( E ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Lipid peroxidation of the AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( F ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( G ) PI staining of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of BSO (500 μM) with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( H ) PI staining images of the indicated AsPC-1 cells treated with or without BSO (500 μM) plus TM sulfate (50 μM) with RSL3 at the indicated concentrations with or without Fer-1 (5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Knockdown, shRNA, Transfection, Western Blot, Staining

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: SLC7A11 is essential for copper deprivation-mediated ferroptosis resistance. ( A ) Vector or flag-SLC7A11 overexpressed AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11). Western blot analysis of lysates from the indicated AsPC-1 cells. ( B ) AsPC-1 cells were transfected with a vector or a plasmid encoding Flag-SLC7A11 and then treated with TM (50 μM) for 24h. Western blot analysis of lysates from the indicated AsPC-1 cells. ( C ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11) with or without transfection with a blank vector or a vector encoding SLC7A11. PI staining of indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of Fer-1 (2.5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( D ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or SLC7A11 siRNA (siSLC7A11) with or without transfection with a blank vector or a vector encoding SLC7A11. PI staining images of indicated AsPC-1 cells treated with RSL3 (0.25 μM) or TM (50 μM) in the presence or absence of Fer-1 (2.5 μM) or DFO (20 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Plasmid Preparation, Transfection, Western Blot, Knockdown, shRNA, Staining

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation upregulates SLC7A11 and activates AMPK-NRF2 axis. ( A ) Volcano plot of NRF2 target genes (shSLC31A1 VS shNC) generated using the data from C. ( B , C ) ChIP analyses of NRF2 binding in the SLC7A11 promoter in NRF2 knockdown AsPC-1 cells (B) or SLC31A1 knockdown AsPC-1 cells (C). ( D-G ) The oxygen consumption rate (OCR) in NC and SLC31A1 knockdown AsPC-1 cells by Seahorse assay (n = 8/group). Quantification of basal respiration (E) and ATP-linked respiration (F), the uncoupler FCCP (maximal) or the electron transport inhibitor antimycin A-baseline (G) in NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 8. Statistical significance was determined using a one-way ANOVA test. ( H ) COX activity level in the NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( I–K ) ATP level in the NC and SLC31A1 knockdown AsPC-1(I), MiaPaCa-2 (J), and CFPAC-1 (K) cells. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( L ) ATP level in the AsPC-1 cells treated with TM (50 μM) or TEPA (1 mM) for 24 h. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test. ( M ) AMP/ATP ratio detected by HPLC in NC and SLC31A1 knockdown AsPC-1 cells. Mean ± SD, n = 3. Statistical significance was determined using an unpaired T test. ( N ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells. ( O ) Western blot analysis of lysates from AsPC-1 cells treated with TM at the indicated concentrations for 24 h. ( P ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with blank vector or vector encoding SLC31A1. ( Q ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with blank vector or of SLC31A1 WT- or SLC31A1 M154A.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Generated, Binding Assay, Knockdown, Activity Assay, Western Blot, shRNA, Transfection, Plasmid Preparation

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation upregulates SLC7A11 through AMPK-NRF2 axis. ( A ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). ( B ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNFE2L2#2). * indicates a non-specific band. ( C ) SLC7A11 mRNA level in NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2#2). ( D ) Western blot analysis of lysates from AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( E ) SLC7A11 mRNA level in AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( F ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells treated with or without ML385 (5 μM) for 24 h. ( G ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. ( H ) SLC7A11 mRNA level in SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Western Blot, Knockdown, shRNA, Transfection, Plasmid Preparation

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation inhibits ferroptosis by the activation of AMPK. ( A ) NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). Propidium iodide (PI) staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( B ) AsPC-1 cells were transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. ( C ) PI staining of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of Compound C (10 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Activation Assay, Knockdown, shRNA, Transfection, Staining

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation inhibits ferroptosis by the activation of AMPK-NRF2-SLC7A11 pathway. ( A ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( B ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( C ) SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( D ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( E ) PI staining images of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of ML385 (5 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( F ) PI staining of SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid, followed by treatment with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Activation Assay, Knockdown, Transfection, shRNA, Staining, Plasmid Preparation

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: SLC31A1 knockdown inhibits ferroptosis in vivo . ( A-D ) Subcutaneous growth of NC and SLC31A1 knockdown AsPC-1 xenografts in nude mice (n = 8) treated with RSL3 (5 mg/kg, i.p., once every other day). ( A ) Tumor growth curves of NC and SLC31A1 knockdown AsPC-1 xenografts in nude mice treated with the indicated agents. Representative images of tumors and tumor weight were shown ( B ). Data are presented as mean ± SD, n = 8 mice per group; Statistical significance was determined using a two-way ANOVA test. ( C ) Total copper content in the tumor sections. Statistical significance was determined using a two-way ANOVA test. ( D and E ) Immunohistochemical staining and relative staining intensity of the indicated proteins in the tumor sections. CC3, cleaved caspase 3. Scale bar: 20 μm. Statistical significance was determined using a two-way ANOVA test.

Article Snippet: SLC31A1 , Cell Signaling Technology , 13086.

Techniques: Knockdown, In Vivo, Immunohistochemical staining, Staining

(a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and CTR1 in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Journal: bioRxiv

Article Title: Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program

doi: 10.64898/2026.03.24.713962

Figure Lengend Snippet: (a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and CTR1 in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Adipocyte-specific and brown adipocyte–specific Ctr1 knockout mice were generated by crossing Ctr1 flox/flox mice with Adipoq -Cre and Ucp1 -Cre transgenic mice (The Jackson Laboratory, strains 028020 and 024670) to produce adipocyte-specific knockout (ACKO; Ctr1 flox/flox ; Adipoq -Cre) and BAT-specific knockout (BCKO; Ctr1 flox/flox ; Ucp1 -Cre) mice, respectively.

Techniques: Western Blot, Control, Expressing, Saline, Two Tailed Test

(a) Immunoblot analysis of ATP7A, CTR1, and CCS in BAT, iWAT, eWAT, and liver from Ctr1 -floxed (Floxed) and adipose-specific Ctr1 knockout (ACKO; Ctr1 fl/fl ; Adipoq -Cre) mice. GAPDH served as a loading control. (b) ICP–MS quantification of Cu levels in tissues from Floxed (n = 5) and ACKO (n = 6) mice. (c) Rectal body temperature in Floxed (n = 6) and ACKO (n = 8) mice during acute cold exposure (CE, 4°C). Food, but not water, was removed at the onset of CE from RT (∼22°C). (d) Kaplan–Meier survival analysis during CE showing reduced survival in ACKO (n = 9) compared with Floxed (n = 8) mice (log-rank test, ****P < 0.0001). Mice were euthanized upon reaching the endpoint criterion (rectal temperature <28°C). (e–g) Indirect calorimetry measurements during gradual transition (∼80 min) from 22°C to 4.5°C. Food, but not water, was removed at the onset of cooling. Core body temperature (e), cold-induced energy expenditure (f), and respiratory exchange ratio (RER) (g) in Floxed (n = 6) and ACKO (n = 6) mice. (h–j) Core body temperature (h), CL-induced energy expenditure (i), and RER (j) before and after intraperitoneal injection of CL (1 mg kg⁻¹) in Floxed (n = 6) and ACKO (n = 6) mice. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Floxed controls. Unless otherwise indicated, experiments were performed in ∼10-week-old male mice.

Journal: bioRxiv

Article Title: Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program

doi: 10.64898/2026.03.24.713962

Figure Lengend Snippet: (a) Immunoblot analysis of ATP7A, CTR1, and CCS in BAT, iWAT, eWAT, and liver from Ctr1 -floxed (Floxed) and adipose-specific Ctr1 knockout (ACKO; Ctr1 fl/fl ; Adipoq -Cre) mice. GAPDH served as a loading control. (b) ICP–MS quantification of Cu levels in tissues from Floxed (n = 5) and ACKO (n = 6) mice. (c) Rectal body temperature in Floxed (n = 6) and ACKO (n = 8) mice during acute cold exposure (CE, 4°C). Food, but not water, was removed at the onset of CE from RT (∼22°C). (d) Kaplan–Meier survival analysis during CE showing reduced survival in ACKO (n = 9) compared with Floxed (n = 8) mice (log-rank test, ****P < 0.0001). Mice were euthanized upon reaching the endpoint criterion (rectal temperature <28°C). (e–g) Indirect calorimetry measurements during gradual transition (∼80 min) from 22°C to 4.5°C. Food, but not water, was removed at the onset of cooling. Core body temperature (e), cold-induced energy expenditure (f), and respiratory exchange ratio (RER) (g) in Floxed (n = 6) and ACKO (n = 6) mice. (h–j) Core body temperature (h), CL-induced energy expenditure (i), and RER (j) before and after intraperitoneal injection of CL (1 mg kg⁻¹) in Floxed (n = 6) and ACKO (n = 6) mice. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Floxed controls. Unless otherwise indicated, experiments were performed in ∼10-week-old male mice.

Techniques: Western Blot, Knock-Out, Control, Injection, Two Tailed Test

(a) Volcano plot of differentially expressed proteins in BAT from Floxed and ACKO mice after 6 h CE (n = 3 per group). Downregulated proteins were subjected to KEGG pathway enrichment analysis (bottom). (b) Immunoblot analysis of OXPHOS complex subunits (ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8), UCP1, and ACTIN in BAT from Floxed and ACKO mice housed at RT or exposed to cold (CE). (c) Oxygen consumption rate (OCR) traces of immortalized pre-brown adipocytes derived from Floxed and ACKO mice treated with vehicle or CL (10 μM). (d) Quantification of respiratory parameters derived from (c), including basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. (e) Relative mRNA expression of Ctr1 and thermogenic genes ( Ucp1 , Prdm16 , Dio2 , Cidea ) in BAT from Floxed and ACKO mice under RT or CE. (f) Representative H&E staining of BAT from Floxed and ACKO mice under RT or CE. Scale bar, 50 μm. (g) TG content in BAT from Floxed and ACKO mice under RT or CE. (h, i) Immunoblot analysis of phosphorylated HSL (Ser660) and total HSL in BAT (h) and iWAT (i) from Floxed and ACKO mice treated with saline or CL (1 mg kg⁻¹, 15 min). (j) Immunoblot analysis of CTR1 and UCP1 in iWAT from Floxed and ACKO mice treated with saline or CL. (k) Relative mRNA expression of Ppargc1a , Ucp1, and Ctr1 in iWAT from Floxed and ACKO mice treated with saline or CL. (l) Cu content in iWAT from Floxed and ACKO mice treated with saline or CL. (m) Representative H&E staining of iWAT from Floxed and ACKO mice treated with saline or CL (1 mg kg⁻¹ day⁻¹, once daily for 10 consecutive days). Scale bar, 50 μm. (n) Relative mtDNA content in iWAT following 10 days of CL treatment. Data are presented as mean ± SEM. Statistical significance for panels (d, e, k, l, n) was determined by one-way ANOVA with Tukey’s post hoc test for each parameter analyzed independently. Groups not sharing a common letter are significantly different (P < 0.05).

Journal: bioRxiv

Article Title: Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program

doi: 10.64898/2026.03.24.713962

Figure Lengend Snippet: (a) Volcano plot of differentially expressed proteins in BAT from Floxed and ACKO mice after 6 h CE (n = 3 per group). Downregulated proteins were subjected to KEGG pathway enrichment analysis (bottom). (b) Immunoblot analysis of OXPHOS complex subunits (ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8), UCP1, and ACTIN in BAT from Floxed and ACKO mice housed at RT or exposed to cold (CE). (c) Oxygen consumption rate (OCR) traces of immortalized pre-brown adipocytes derived from Floxed and ACKO mice treated with vehicle or CL (10 μM). (d) Quantification of respiratory parameters derived from (c), including basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. (e) Relative mRNA expression of Ctr1 and thermogenic genes ( Ucp1 , Prdm16 , Dio2 , Cidea ) in BAT from Floxed and ACKO mice under RT or CE. (f) Representative H&E staining of BAT from Floxed and ACKO mice under RT or CE. Scale bar, 50 μm. (g) TG content in BAT from Floxed and ACKO mice under RT or CE. (h, i) Immunoblot analysis of phosphorylated HSL (Ser660) and total HSL in BAT (h) and iWAT (i) from Floxed and ACKO mice treated with saline or CL (1 mg kg⁻¹, 15 min). (j) Immunoblot analysis of CTR1 and UCP1 in iWAT from Floxed and ACKO mice treated with saline or CL. (k) Relative mRNA expression of Ppargc1a , Ucp1, and Ctr1 in iWAT from Floxed and ACKO mice treated with saline or CL. (l) Cu content in iWAT from Floxed and ACKO mice treated with saline or CL. (m) Representative H&E staining of iWAT from Floxed and ACKO mice treated with saline or CL (1 mg kg⁻¹ day⁻¹, once daily for 10 consecutive days). Scale bar, 50 μm. (n) Relative mtDNA content in iWAT following 10 days of CL treatment. Data are presented as mean ± SEM. Statistical significance for panels (d, e, k, l, n) was determined by one-way ANOVA with Tukey’s post hoc test for each parameter analyzed independently. Groups not sharing a common letter are significantly different (P < 0.05).

Techniques: Western Blot, Derivative Assay, Expressing, Staining, Saline

(a) Rectal body temperature of Ctr1 -floxed and ACKO mice treated with vehicle (Veh) or elesclomol (ES) during acute cold exposure (4°C). ES treatment improved cold tolerance (two-way ANOVA, treatment effect *P < 0.05; n = 3–4 per group). (b) Representative H&E staining of BAT from Ctr1-floxed and ACKO mice treated with Veh or ES under CE. Scale bar, 50 μm. (c) Immunoblot analysis of UCP1, OXPHOS complex subunits (ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8), CCS, and GAPDH in BAT from Ctr1 -floxed and ACKO mice treated with Veh or ES. (d, e) Mitochondrial respiration analysis in immortalized pre-brown adipocytes derived from Ctr1 -floxed and ACKO mice treated with ES (10 nM) for 24 h. (d) Oxygen consumption rate (OCR) traces. (e) Quantification of basal respiration, ATP-linked respiration, proton leak, spare respiratory capacity, maximal respiration, and non-mitochondrial respiration. Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post hoc test unless otherwise indicated. Groups not sharing a common letter are significantly different (P < 0.05, one-way ANOVA with Tukey’s post hoc test). (f) Working model illustrating the crosstalk between CTR1-mediated Cu import and adaptive thermogenesis , . Upon cold exposure, norepinephrine (NE) activates β3-adrenergic receptor (β3-AR) signaling, increasing cAMP levels and activating protein kinase A (PKA). PKA promotes thermogenic gene expression (via PGC-1α and CREB) and phosphorylates hormone-sensitive lipase (HSL) to stimulate lipolysis. Released free fatty acids (FFAs) activate UCP1 and provide substrates for mitochondrial oxidative phosphorylation (OXPHOS). CTR1-dependent Cu import supports mitochondrial OXPHOS capacity and thermogenic output, whereas Cu delivery by elesclomol (ES) partially restores oxidative function in Ctr1 -deficient adipocytes. The model highlights outstanding questions, including whether β3-AR signaling regulates CTR1 activity and whether mechanisms exist that prioritize Cu delivery to mitochondria during thermogenic activation.

Journal: bioRxiv

Article Title: Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program

doi: 10.64898/2026.03.24.713962

Figure Lengend Snippet: (a) Rectal body temperature of Ctr1 -floxed and ACKO mice treated with vehicle (Veh) or elesclomol (ES) during acute cold exposure (4°C). ES treatment improved cold tolerance (two-way ANOVA, treatment effect *P < 0.05; n = 3–4 per group). (b) Representative H&E staining of BAT from Ctr1-floxed and ACKO mice treated with Veh or ES under CE. Scale bar, 50 μm. (c) Immunoblot analysis of UCP1, OXPHOS complex subunits (ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8), CCS, and GAPDH in BAT from Ctr1 -floxed and ACKO mice treated with Veh or ES. (d, e) Mitochondrial respiration analysis in immortalized pre-brown adipocytes derived from Ctr1 -floxed and ACKO mice treated with ES (10 nM) for 24 h. (d) Oxygen consumption rate (OCR) traces. (e) Quantification of basal respiration, ATP-linked respiration, proton leak, spare respiratory capacity, maximal respiration, and non-mitochondrial respiration. Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post hoc test unless otherwise indicated. Groups not sharing a common letter are significantly different (P < 0.05, one-way ANOVA with Tukey’s post hoc test). (f) Working model illustrating the crosstalk between CTR1-mediated Cu import and adaptive thermogenesis , . Upon cold exposure, norepinephrine (NE) activates β3-adrenergic receptor (β3-AR) signaling, increasing cAMP levels and activating protein kinase A (PKA). PKA promotes thermogenic gene expression (via PGC-1α and CREB) and phosphorylates hormone-sensitive lipase (HSL) to stimulate lipolysis. Released free fatty acids (FFAs) activate UCP1 and provide substrates for mitochondrial oxidative phosphorylation (OXPHOS). CTR1-dependent Cu import supports mitochondrial OXPHOS capacity and thermogenic output, whereas Cu delivery by elesclomol (ES) partially restores oxidative function in Ctr1 -deficient adipocytes. The model highlights outstanding questions, including whether β3-AR signaling regulates CTR1 activity and whether mechanisms exist that prioritize Cu delivery to mitochondria during thermogenic activation.

Techniques: Staining, Western Blot, Derivative Assay, Gene Expression, Phospho-proteomics, Activity Assay, Activation Assay

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