Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Schematic of copper transport between the blood and brain in controls (A) and schizophrenia (B). Stars show copper. CTR1, white arrows; ATP7A, black arrows; ATP7B, striped arrows; BBB, blood brain barrier; BCB, blood cerebrospinal barrier. Thinner arrows indicate decreased protein levels.

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques:

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Protein levels of ATP7A (A-B), CTR1 (C-D), dysbindin 1A, 1B/C (E-F) and ATP7B (G-H) for analysis of diagnostic group and treatment status. Error bars represent standard deviation. Significant omnibus ANOVA results are: N-terminal ATP7A: p=0.02; C-terminal ATP7A: p=0.01; transmembrane CTR1: p=0.001. Significant ANOVAs were followed by post hoc tests illustrated by the following: *: p<0.05; **: p<0.01; ***: p<0.001.

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques: Diagnostic Assay, Standard Deviation

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Numerical labeling of amino acid ranges are shown for each protein segment. Dotted boxes indicate antibody-specific epitopes. A. ATP7A protein structure. N-terminal antibody specifically binds to 225-273aa; C-terminal antibody binds to 1403-1452aa. B. CTR1 protein structure. Extracellular CTR1 antibody binds to 19-68aa; transmembrane CTR1 antibody specifically binds to 140-190aa. C. Intracellular diagram of copper chaperones and enzymes and schizophrenia-related alterations. Copper enters neurons after transport from astrocytes via CTR1 and is immediately bound by MT/ GSH. MT/GSH then delivers copper to chaperone ATOX1 or the TGN where it is delivered to other metalloenzymes (e.g., SCO1 → COX) via ATP7A. The SN exhibits more ATOX1 than other brain regions. SCO1 is a copper-requiring enzyme involved in the last step of the electron transport chain of ATP synthesis. SCO1, MT/GSH, COX, ATP, ATP7A and CTR1 are all downregulated or altered in schizophrenia. Abbreviations: MT, metallothionein; GSH, glutathione; APD, antipsychotic drug; SN, substantia nigra; COX, cytochrome c oxidase. References: 1) The present paper; 2) ; 3) ; 4) ; 5) ; 6) ; ; 7) ; 8) ; 9) ; 10) ; and 11) .

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques: Labeling

Journal: Muscle & nerve

Article Title: Amyotrophic Lateral Sclerosis and swim training affect copper metabolism in skeletal muscle in a mouse model of disease.

doi: 10.1002/mus.28237

Figure Lengend Snippet: FIGURE 2 Effects of amyotrophic lateral sclerosis (ALS) disease progression and swim training on ATP7a, CTR1, and DMT1 protein level in skeletal muscle. ATP7a (A), CTR1 (B), and DMT1 (C) levels were measured in mice skeletal muscle. The cropped blots were used in the figure. Full-length blots are shown in Figures S2–S4. There were significant differences between the groups: ap < .05, bp < .01, dp < .001 versus ALS BEFORE group, Ap < .05, Bp < .01 versus ALS ONSET untrained group of mice, **p < .01, ***p < .001 versus wild type (WT) group of mice (Tukey's post hoc test), #p < .05, ##p < .01 between the indicated groups (Student t-test). The data are presented as the means ± SD (n = 6 in each group).

Article Snippet: Next, the membranes were rinsed three times in 1 TBST for 5 min, incubated with primary antibodies dissolved in blocking buffer with gentle shaking, and left overnight at 4 C. The following rabbit polyclonal antibody was used: CTR1/ SLC31A1 (Cat. No. 13086, Cell Signaling, Danvers, MA, USA, 1:1000), DMT1/anti-SLC11A1 (Cat. No. FNab07905, Fine Test, Wuhan, China, 1:500), anti-ATP7a (Cat. No. ab 125,137, Abcam, Cambridge, UK, 1:1000), and rabbit monoclonal antibody; CCS/CCS antibody (Cat. No. ab137131, Abcam, Cambridge, UK, 1:1000).

Techniques: Biomarker Discovery

Journal: Nature Communications

Article Title: Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis

doi: 10.1038/s41467-020-14698-y

Figure Lengend Snippet: a Western blots analysis for copper trafficking-associated proteins from lysates of mouse colon epithelial organoids treated with different inflammatory cytokines for 8 h. Three independent experiments were done and representative blots were shown. Untr untreated. b IL-17 primed or unprimed mouse colon organoids were stained for STEAP4 (St4), E-cadherin (E-cad) and Ctr1 and integrin α. Scale bar, 100 µm. c SOD enzyme activity recovery from TTM inhibition in wild-type (WT) mouse organoids with or without 12 h of IL-17 priming. d SOD enzyme activity recovery in Steap4 wild-type (WT) and knockout (KO) organoids. e , f Fluorometric detection of intracellular copper in primary mouse colon organoids ( e ) or Ls174t cells ( f ). CF4 copper Fluor-4 probe, Ctrl CF4 control probe. Scale bar,100 µm. n = 5 biologically independent cell cultures. g Fluorometric detection of intracellular copper in Steap4 WT and Ctr1 knockdown cells. Cells were treated with or without IL-17 for 12 h and copper were supplemented into the medium for 6 h in all the groups. Scale bar,100 µm. n = 5 biologically independent cell cultures. h Atomic absorption spectroscopy measurement of copper content in Steap4 WT and KO Ls174t cells after copper addition. Cells were primed with or without IL-17 for 12 h and then 10 µM Cu(II) were supplemented into the medium of the indicated group for 6 h. n = 5 biologically independent cell cultures. i Copper content in cytosolic and mitochondrial fraction in Steap4 WT and KO cells, respectively. Cells were treated the same as described in panel ( h ). n = 5 biologically independent cell cultures. Results were normalized to cell numbers. All data show mean ± SEM. Two-tailed Student’s t test was performed for panel e (** P = 0.0057; *** P = 0.0004; **** P < 0.0001), f (*** P = 0.0004; **** P < 0.0001), g (* P = 0.015; ** P = 0.0027; *** P = 0.0004), h (* P = 0.0383; ** P = 0.0022; *** P = 0.0009), i (*** P = 0.0003; **** P < 0.0001; n.s., not significant). Data represent three independent experiments with similar results.

Article Snippet: The following primary antibodies were used for staining presented in the study: Ki67 (Cell Signaling Technology 12202, 1:200), Cleaved Caspase 3 (Cell Signaling Technology 9661, 1:300), STEAP4 (Proteintech, 11944-1-AP,1:100), IL-17A (R&D Systems, MAB317, 1:300), DYKDDDDK (Cell Signaling, 14793S, 1:300), Ctr1 (Novus Biologicals NBP2-36573, 1:100), TUNEL assay (Sigma, 11684795910).

Techniques: Western Blot, Staining, Activity Assay, Inhibition, Knock-Out, Control, Knockdown, Atomic Absorption Spectroscopy, Two Tailed Test

Journal: BMC Cancer

Article Title: Matrigel-based organoid culture of malignant mesothelioma reproduces cisplatin sensitivity through CTR1

doi: 10.1186/s12885-023-10966-4

Figure Lengend Snippet: Matrigel mediates high sensitivity to cisplatin. a Phase-contrast images of m5 MM-organoids before and after cisplatin (100 μg /ml, 24 h) treatment. The majority of MM organoids die within 24 h after cisplatin treatment. b , c Cell viability of m5/m107 MM-organoids and 2D MM cell lines in different cisplatin dosage (0.5–200 μg/ml, 24 h). IC50 values (concentration responsible for 50% cell growth inhibition) are shown in each line. Green lines, MM-organoid; black lines, 2D MM-cell lines. d Slc31a1 ( Ctr1 ) expression level in MM-organoids and 2D MM cell lines from RNA-sequence data. e H&E staining (upper) and immunofluorescent images from m5 MM primary tissue sample, SC injection and organoids. CTR1 (Alexa488, green); nuclear staining (Hoechst33342, blue; scale bar = 20 μm). f Immunoblot analysis of m5/m107 MM-organoids (3D) and 2D MM cell lines (2D); g, glycosylation (30–50 kDa); CTR1 (25 kDa); t, truncate (15 kDa). g , h Enrichment analysis using DAVID indicates significant activation of protein glycosylation and lipidation pathways between mesothelioma 2D- and 3D-cultures

Article Snippet: EMEM (Wako, 051–07,615, Osaka, Japan), FBS (Biowest, S1810-500; Nuaillé, France), Tet system Approved FBS (TaKaRa, 631,107, Shiga, Japan), Autofluorescence Quenching Kit (Vector, SP-8400, Burlingame, CA), AurumTM Total RNA Mini Kit (BioRad, 64,337,836, Hercules, CA), NEBNext Ultra II Directional RNA Library prep kit for Illumina (BioLabs, E7760S, Boston, MA), NEBNext Multiplex Oligos for Illumina (BioLabs, E6440S), anti-CTR1 (CST, #13,086, Danvers, MA, Novus, #NBP2-36,573), anti-Flag (MBL, PM020, M185-3L, Nagoya, Japan), anti-golgin97 (CST, #13,192), anti-mouse mesothelin (IBL, #28,127, Gunma, Japan), anti-WT1 (Thermo Fischer Scientific, MA5-32,215), anti-ITGβ1 (CST, #9699), anti-IGFR (Proteintech, 20,254–1-AP, Chicago, IL), anti-β-catenin (CST, #8480 T), anti-GFP (MBL life science, #598), anti-mouse IgG Alexa488 (ThermoFischer Scientific, A-11108), anti-rabbit IgG Alexa 568 (Thermo Fischer Scientific, A-11011), anti-rabbit IgG Alexa 633 (Thermo Fischer Scientific, A-21070) CF®568 Phalloidin (biotinum,00064 T), Receptor Tyrosine Kinase Antibody Sampler Kit (CST, #42,344), Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (CST, #9782), anti-β-actin (Sigma Aldrich, P2120, St. Louis, MO) Advanced F12/DMEM (Thermo Fischer Scientific, 12,634,010), recombinant Murine R-Spondin-1 (PeproTech Inc., 315–32, Cranbury, NJ), recombinant Murine Noggin (PeproTech Inc., 250–38), recombinant Murine EGF (PeproTech Inc., 315–09), recombinant Wnt3A (PeproTech Inc., 315–20-2ug), recombinant FGF-basic (PeproTech Inc., 450–33), B-27™ Supplement (50x) (Thermo Fischer Scientific, 17,504,044), N-2 Supplement (100x) (ThermoFischer Scientific, 17,502,048), A8301 (Peprotech Inc., 9,094,360), N-Acetyl-L-cysteine (Wako, 017–05,131) CTS™ GlutaMAX™-I Supplement (ThermoFischer Scientific, A1286001), CultureSure® Y-27632 (Wako, 030–24,021) and Corning® Matrigel® Growth Factor Reduced (GFR) (Corning, 354,230) were used.

Techniques: Concentration Assay, Inhibition, Expressing, Sequencing, Staining, Injection, Western Blot, Activation Assay

Journal: BMC Cancer

Article Title: Matrigel-based organoid culture of malignant mesothelioma reproduces cisplatin sensitivity through CTR1

doi: 10.1186/s12885-023-10966-4

Figure Lengend Snippet: CTR1 localization regulated by extracellular stiffness and glycosylation. a Immunoblot analysis of CTR1 in m5-2D MM cell line, comparing mock and overexpression (OE) of flag-CTR1 group; g, glycosylation (30–50 kDa); CTR1 (25 kDa); t: truncate (15 kDa). b Schematic images of the culture system. High stiffness culture corresponds to direct plating whereas low stiffness culture corresponds to plating on the Matrigel layer (scale bar = 100 μm). c Immunoblot analysis of CTR1. Matrigel, Matrigel layer in the legend (b). MβCD (10 μM, 1 h); g, glycosylation (30–50 kDa); CTR1 (25 kDa); t: truncate (15 kDa). d Immunofluorescent images from m5 Flag-CTR1 stably expressed cell line. Left images; cells were cultured on glass slip as high stiffness culture. Right images; cells were cultured on Matrigel layer as a low stiffness culture. Flag-CTR1 (Alexa 488, green), F-actin (CF 568, magenta) and nuclear staining (Hoechst33342, blue; scale bar, 5 μm). e , f Immunoblot analysis of m5 Flag-CTR1 stably expressed cell after treatment of TM (tunicamycin) or BP (2-bromopalmitate) for 9 h. g, glycosylation (30–50 kDa); CTR1 (25 kDa); t, truncate (15 kDa). g Immunofluorescent images from m5 Flag-CTR1 stably expressed cells with after treatment of TM or BP for 9 h. Flag-CTR1 (Alexa 488, green), F-actin (CF 568, magenta) and nuclear staining (Hoechst33342, blue; scale bar: 20 μm). Refer to text for details

Article Snippet: EMEM (Wako, 051–07,615, Osaka, Japan), FBS (Biowest, S1810-500; Nuaillé, France), Tet system Approved FBS (TaKaRa, 631,107, Shiga, Japan), Autofluorescence Quenching Kit (Vector, SP-8400, Burlingame, CA), AurumTM Total RNA Mini Kit (BioRad, 64,337,836, Hercules, CA), NEBNext Ultra II Directional RNA Library prep kit for Illumina (BioLabs, E7760S, Boston, MA), NEBNext Multiplex Oligos for Illumina (BioLabs, E6440S), anti-CTR1 (CST, #13,086, Danvers, MA, Novus, #NBP2-36,573), anti-Flag (MBL, PM020, M185-3L, Nagoya, Japan), anti-golgin97 (CST, #13,192), anti-mouse mesothelin (IBL, #28,127, Gunma, Japan), anti-WT1 (Thermo Fischer Scientific, MA5-32,215), anti-ITGβ1 (CST, #9699), anti-IGFR (Proteintech, 20,254–1-AP, Chicago, IL), anti-β-catenin (CST, #8480 T), anti-GFP (MBL life science, #598), anti-mouse IgG Alexa488 (ThermoFischer Scientific, A-11108), anti-rabbit IgG Alexa 568 (Thermo Fischer Scientific, A-11011), anti-rabbit IgG Alexa 633 (Thermo Fischer Scientific, A-21070) CF®568 Phalloidin (biotinum,00064 T), Receptor Tyrosine Kinase Antibody Sampler Kit (CST, #42,344), Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (CST, #9782), anti-β-actin (Sigma Aldrich, P2120, St. Louis, MO) Advanced F12/DMEM (Thermo Fischer Scientific, 12,634,010), recombinant Murine R-Spondin-1 (PeproTech Inc., 315–32, Cranbury, NJ), recombinant Murine Noggin (PeproTech Inc., 250–38), recombinant Murine EGF (PeproTech Inc., 315–09), recombinant Wnt3A (PeproTech Inc., 315–20-2ug), recombinant FGF-basic (PeproTech Inc., 450–33), B-27™ Supplement (50x) (Thermo Fischer Scientific, 17,504,044), N-2 Supplement (100x) (ThermoFischer Scientific, 17,502,048), A8301 (Peprotech Inc., 9,094,360), N-Acetyl-L-cysteine (Wako, 017–05,131) CTS™ GlutaMAX™-I Supplement (ThermoFischer Scientific, A1286001), CultureSure® Y-27632 (Wako, 030–24,021) and Corning® Matrigel® Growth Factor Reduced (GFR) (Corning, 354,230) were used.

Techniques: Western Blot, Over Expression, Stable Transfection, Cell Culture, Staining

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: High expressions of Ctr1 mRNA and protein in ESCC tissues and cells. A: Representative image of Ctr1 high expression in ESCC tissues by ISH and IHC; ISH and IHC assay for Ctr1 mRNA and protein expressions in 108 cases of ESCC tissues and paired normal esophageal epithelial tissues, Bar=100 μm. B: Semi-quantitative RT-PCR detection for Ctr1 mRNA expression in randomly selected 10 cases of ESCC tissues and paired normal tissues using Ctr1 specific primers. C: Statistical assay for Ctr1 relative level in ESCC tissues and paired normal tissues, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, compared para-carcinoma tissues. D: qRT-PCR assay for Ctr1 mRNA expression in ESCC cell lines (Kyse70, Eca109, and Kyse450 cells), ** P <0.01 and *** P <0.001, compared with Het-1A cell. E: Western blot analysis for Ctr1 protein expression in ESCC cell lines (Kyse70, Eca109, and Kyse450 cells), and β-actin was used as loading control. F: Relative Ctr1 protein level in various ESCC cell lines (Kyse70, Eca109, and Kyse450 cells), ** P <0.01, compared with Het-1A cell.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

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

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: The expressions of Ctr1 mRNA and protein in ESCC tissues and normal tissues.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques:

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: The associations of Ctr1 mRNA and protein expressions with clinicopathological features in ESCC.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques:

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: The Ctr1 downregulation reduces the sensitivity of cisplatin in ESCC cells. A: Ctr1 siRNA significantly suppressed Ctr1 mRNA expression at 24 h, 48 h and 72 h in various ESCC cells (Eca109, Kyse70 and Kyse450), si-Con and si-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by Lipofectamine™ 2000, and Semi-quantitative RT-PCR was used to determine the relative level of Ctr1 at 24 h, 48 h and 72 h after transfection, ** P <0.01 and *** P <0.001, compared with control group and si-Con group. B: Western blot was performed to investigate the Ctr1 protein expression at 24 h, 48 h and 72 h after transfection with si-Ctr1 and si-Con in different ESCC cells (Eca109, Kyse70 and Kyse450), and β-actin was employed as a loading control. C: The relative level of Ctr1 was counted using the rate of Ctr1 protein level to β-actin level in diverse ESCC cells (Eca109, Kyse70 and Kyse450), * P <0.05, ** P <0.01 and *** P <0.001, compared with control group and si-Con group. D: The Ctr1 downregulation reduced the cytotoxicity of cisplatin in distinct ESCC cells. ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and CCK-8 was used to determine cell viability, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, indicating statistical significance, compared with si-Con.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Control, Western Blot, CCK-8 Assay

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: Ctr1 depletion prominently repressed cell apoptosis evoked by cisplatin in ESCC cells. A: Flow cytometry assay for cell apoptosis in different ESCC cells treated with si-Con, si-Con plus cisplatin, si-Ctr1 as well as si-Ctr1 plus cisplatin; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Flow cytometry was employed to determine cell apoptosis. B: Statistical assay for apoptotic cell numbers in different ESCC cells treated with si-Con, si-Con plus cisplatin, si-Ctr1 as well as si-Ctr1 plus cisplatin, *** P <0.001 and **** P <0.0001, compared with si-Con plus cisplatin group. C: Ctr1 depletion combined cisplatin significantly suppressed the activity of Caspase-3 induced by cisplatin plus si-Con; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Caspase-3 activity kit was utilized to determine the activity of Caspase-3, *** P <0.001, compared with si-Con plus cisplatin group.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques: Flow Cytometry, Transfection, Activity Assay

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: The Ctr1 downregulation meliorated cell migration and invasion abilities inhibited by cisplatin in number of ESCC cells. A: Transwell assay for cell migration in various ESCC cells treated with si-Con, si-Con plus cisplatin, si-Ctr1 and si-Ctr1 plus cisplatin; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Transwell chamber without Matrigel was employed to determine cell migration. B: Statistical assay for migratory cell numbers in different ESCC cells treated with si-Con, si-Con plus cisplatin, si-Ctr1 and si-Ctr1 plus cisplatin, *** P <0.001, compared with si-Con plus cisplatin group. C: Transwell assay for cell invasion in varied ESCC cells treated with si-Con, si-Con plus cisplatin, si-Ctr1 and si-Ctr1 plus cisplatin; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Transwell chamber with Matrigel was used to determine cell invasion. D: Statistical assay for invasive cell numbers in a variety of ESCC cells treated with si-Con, si-Con plus cisplatin, si-Ctr1 and si-Ctr1 plus cisplatin, *** P <0.001, compared with si-Con plus cisplatin group.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques: Migration, Transwell Assay, Transfection

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: The Ctr1 upregulation potentiated the killing efficacy of cisplatin in ESCC cells. A: pcDNA3.1-Ctr1 remarkablely enhanced the Ctr1 mRNA level at 48 h in various ESCC cells (Eca109, Kyse70 and Kyse450), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by Lipofectamine™ 2000, and Semi-quantitative RT-PCR was used to determine the relative level of Ctr1 at 48 h after transfection,** P <0.01 and *** P <0.001, compared with control group and pcDNA3.1 group. B: Western blot was performed to investigate the Ctr1 protein expression in a number of ESCC cells 48 h after transfection with pcDNA3.1 and pcDNA3.1-Ctr1, and β-actin was used as a loading control. C: The relative level of Ctr1 protein in untreated ESCC cells, ESCC cells transfected with pcDNA3.1 and pcDNA3.1-Ctr1, ** P <0.01, compared with control group and pcDNA3.1 group. D: CCK-8 was used to examine the cell proliferative ability in different concentration of cisplatin with pcDNA3.1 or pcDNA3.1-Ctr1, ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and CCK-8 was used to determine cell viability, ** P <0.01, *** P <0.001 and **** P <0.0001, indicating statistical significance, compared with pcDNA3.1.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques: Transfection, Quantitative RT-PCR, Control, Western Blot, Expressing, CCK-8 Assay, Concentration Assay

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: Ctr1 upregulation combined with cisplatin displayed the synergistic role in the induction of cell apoptosis in ESCC cells. A: Flow cytometry assay for cell apoptosis in different ESCC cells treated with pcDNA3.1, pcDNA3.1 plus cisplatin, pcDNA3.1-Ctr1 and pcDNA3.1-Ctr1 plus cisplatin, ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Flow cytometry was employed to determine cell apoptosis. B: Statistical assay for apoptotic cell numbers in different ESCC cells treated with pcDNA3.1, pcDNA3.1 plus cisplatin, pcDNA3.1-Ctr1 and pcDNA3.1-Ctr1 plus cisplatin, ** P <0.01 and *** P <0.001, compared with pcDNA3.1 plus cisplatin group. C: pcDNA3.1-Ctr1 combined cisplatin significantly promoted the activity of Caspase-3 induced by cisplatin plus pcDNA3.1; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Caspase-3 activity kit was utilized to determine the activity of Caspase-3, ** P <0.01 and *** P <0.001, compared with pcDNA3.1 plus cisplatin group.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques: Flow Cytometry, Transfection, Activity Assay

Journal: Translational Oncology

Article Title: Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

doi: 10.1016/j.tranon.2023.101626

Figure Lengend Snippet: Ctr1 upregulation exerted the synergistic role in the repression of cell migration and invasion with cisplatin in ESCC cells. A: Transwell assay for cell migration in various ESCC cells treated with pcDNA3.1, pcDNA3.1 plus cisplatin, pcDNA3.1-Ctr1 and pcDNA3.1-Ctr1 plus cisplatin; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Transwell chamber without Matrigel was employed to determine cell migration. B: Statistical assay for migratory cell numbers in different ESCC cells treated with pcDNA3.1, pcDNA3.1 plus cisplatin, pcDNA3.1-Ctr1 and pcDNA3.1-Ctr1 plus cisplatin, ** P <0.01 and **** P <0.0001, compared with pcDNA3.1 plus cisplatin group. C: Transwell assay for cell invasion in varied ESCC cells treated with pcDNA3.1, pcDNA3.1 plus cisplatin, pcDNA3.1-Ctr1 and pcDNA3.1-Ctr1 plus cisplatin; ESCC cells (Eca109, Kyse70 and Kyse450) were harvested at 48 h after transfection, different doses of cisplatin was applied to ESCC cells above, and Transwell chamber with Matrigel was employed to determine cell invasion. D: Statistical assay for invasive cell numbers in a variety of ESCC cells treated with pcDNA3.1, pcDNA3.1 plus cisplatin, pcDNA3.1-Ctr1 and pcDNA3.1-Ctr1 plus cisplatin, ** P <0.01 and *** P <0.001, compared with pcDNA3.1 plus cisplatin group.

Article Snippet: Subsequently, primary antibody against Ctr1 (GeneTex company, USA) diluted 1:200 was incubated with tissue slides overnight at 4 °C.

Techniques: Migration, Transwell Assay, Transfection