Journal: bioRxiv

Article Title: Altered Copper Transport in Oxidative Stress-Dependent Brain Endothelial Barrier Dysfunction Associated with Alzheimer’s Disease

doi: 10.1101/2024.08.28.610108

Figure Lengend Snippet: A and B . hBMEC were pretreated with thiol antioxidant N-Acetyl Cysteine (NAC)(10 mM) for 1 h or Cu chelator, TTM (20 nM) for 24 hr (A), or transfected with siRNAs for CTR1 or scrambled control (30 nM)(B). These ECs stimulated with 20 μM Aβ42 for indicated times were used for TEER assay to measure EC permeability. Data analysis are shown in the right panel.

Article Snippet: Control siRNA and CTR1 siRNA were obtained from Ambion and Invitrogen respectively.

Techniques: Transfection, Control, Permeability

Journal: bioRxiv

Article Title: Altered Copper Transport in Oxidative Stress-Dependent Brain Endothelial Barrier Dysfunction Associated with Alzheimer’s Disease

doi: 10.1101/2024.08.28.610108

Figure Lengend Snippet: A and B, hBMECs stimulated with Aβ42 (20 μM) for the indicated time (0-16hr) (A), or transduced with adenovirus expressing catalase (Ad. Catalase) stimulated with Aβ42 (20 μM) for 16hr, or exposed to scrambled Aβ 42 (Scr. Aβ42) (20 μM) for 4 hr were used to measure DCF fluorescence (ROS production) ( B ). Representative images for DCF fluorescence with DAPI (blue) staining (Left) and quantification of DCF fluorescence intensity expressed as a fold change from the basal control (Right) are shown. Data are mean ± SEM (n=3). ****p<0.0001. C and D, hBMECs pretreated with TTM (20 nM) for 24 h ( C ), or transfected with siRNAs for CTR1 or control (30 nM) ( D ) were stimulated with 20 μM Aβ42 for 4 hr. These ECs were used for DCF fluorescence assay to measure ROS production. Representative images for DCF fluorescence with DAPI (blue) staining (Left) and quantification of DCF fluorescence intensity expressed as a fold change from the basal control (Right) are shown. Data are mean ± SEM (n=3) #p<0.001.

Article Snippet: Control siRNA and CTR1 siRNA were obtained from Ambion and Invitrogen respectively.

Techniques: Transduction, Expressing, Fluorescence, Staining, Control, Transfection

Journal: bioRxiv

Article Title: Altered Copper Transport in Oxidative Stress-Dependent Brain Endothelial Barrier Dysfunction Associated with Alzheimer’s Disease

doi: 10.1101/2024.08.28.610108

Figure Lengend Snippet: A,B,C. hBMECs pretreated with TTM (20 nM for 24 h) or antioxidant NAC (10 mM for 1 h)( A ), or transfected with siRNAs for CTR1 or control (30 nM)( B ) were stimulated with Aβ42. In parallel, hBMECs were stimulated with scrambled Aβ42 (20 μM) ( C ). Lysates from these cells were used for western blotting with anti-VE-Cadherin (VE-Cad)-pY658 or anti-VE-Cad or β-actin (loading control) antibodies. Bar graphs represent the fold changes from the basal control. Data are mean ± SEM (n=3). ***p<0.001, ****p<0.0001.

Article Snippet: Control siRNA and CTR1 siRNA were obtained from Ambion and Invitrogen respectively.

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: Altered Copper Transport in Oxidative Stress-Dependent Brain Endothelial Barrier Dysfunction Associated with Alzheimer’s Disease

doi: 10.1101/2024.08.28.610108

Figure Lengend Snippet: Aβ42 induces Cu dysregulation through upregulation of CTR1 and downregulation of ATP7A/ATP7B, which increases intracellular Cu and promotes ROS production and ROS-dependent tyrosine phosphorylation of VE-Cadherin, which causes brain endothelial barrier dysfunction, leading to loss of BBB integrity, resulting in progression of AD pathology.

Article Snippet: Control siRNA and CTR1 siRNA were obtained from Ambion and Invitrogen respectively.

Techniques:

Journal: Life

Article Title: Cytotoxic Activity of the Red Grape Polyphenol Resveratrol against Human Prostate Cancer Cells: A Molecular Mechanism Mediated by Mobilization of Nuclear Copper and Generation of Reactive Oxygen Species

doi: 10.3390/life14050611

Figure Lengend Snippet: The growth rate of MCF-10A-Cu cells (non-tumorigenic MCF-10A cells grown in a medium containing 25 µM CuCl 2 ) was noticeably reduced on exposure to resveratrol after the suppression of CTR1 . The MCF-10A+Cu cells were initially exposed to resveratrol or specific siRNA against CTR1 (si CTR1 ) for 48 h, followed by treatment with specified doses of resveratrol after 24 h. The values presented represent the standard error (±S.E.) of three separate studies. * p value < 0.05 when compared to control.

Article Snippet: Santa Cruz Biotechnology, Inc. was approached regarding CTR1 -specific siRNA.

Techniques: 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: 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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

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: Control siRNA (si-Con) (Santa Cruz company, USA), Ctr1 siRNA (si-Ctr1) (Santa Cruz company, USA), pcDNA3.1 and pcDNA3.1-Ctr1 were transfected to Eca109, Kyse70 and Kyse450 cells by LipofectamineTM 2000 (Invitrogen Life Technologies, Carslbad, CA, USA) according to manufacturer's instruction.

Techniques: Migration, Transwell Assay, Transfection

Journal: PLoS ONE

Article Title: Upregulated Copper Transporters in Hypoxia-Induced Pulmonary Hypertension

doi: 10.1371/journal.pone.0090544

Figure Lengend Snippet: A–C: Western blot analysis of mouse CTR1 (A), pro-LOX (B) and HIF-1α (C) in total and membrane proteins extracted from whole-lung lung tissues of normoxic control mice (Nor, n = 5) and chronically hypoxic (Nor, n = 5) mice. Proteins from Nor and Hyp mouse lungs were solubilized in 3% DDM/1× RIPA buffer and utilized for Western blot analysis using antibodies specific for mouse CTR1, pro-LOX, and HIF-1α. β-actin or β-tubulin was used as a loading control. D: Summarized data (mean±SE) showing protein expression levels of CTR1, pro-LOX and HIF-1α in lungs tissues isolated from Nor and Hyp mice. The band intensity was quantitated with ImageJ software, normalized with respect to the loading control, and then shown relative to control (% of Nor). ** P <0.01 vs. Nor.

Article Snippet: siRNA oligos for HIF-1α, HIF-2α, and CTR1 knockdowns were purchased from Ambion (prod. nos. s6539, s4700, and s3377), and used at final concentration of 100 pmol per 6 well-plate for 48–72 hrs. siRNA was introduced into the cells using RNA Max Lipofectamine reagent (Invitrogen).

Techniques: Western Blot, Expressing, Isolation, Software

Journal: PLoS ONE

Article Title: Upregulated Copper Transporters in Hypoxia-Induced Pulmonary Hypertension

doi: 10.1371/journal.pone.0090544

Figure Lengend Snippet: A: Real-time RT-PCR analysis on HIF-1α ( a ), HIF-2α ( b ) and CTR1 ( c ) in hypoxic PASMC treated with (50–200 pmol) or without (0 pmol) siRNA specifically targeting HIF-1α, HIF-2α and CTR1, respectively. Data are shown in mean±SE. *** P <0.01 vs. control hypoxic cells (0-pmol siRNA). B: Real-time RT-PCR analysis on human CTR1 ( a ), ATP7A ( b ), HIF-1α ( c ) and HIF-2α ( d ) in hypoxic PASMC treated with 100-pmol scrambled siRNA (Control-siRNA, open bars), HIF-1α-siRNA (solid bars), HIF-2α-siRNA (light grey bars), and CTR1-siRNA (dark grey bars), respectively. *** P <0.01 vs. hypoxic cells treated with scrambled siRNA (Control-siRNA).

Article Snippet: siRNA oligos for HIF-1α, HIF-2α, and CTR1 knockdowns were purchased from Ambion (prod. nos. s6539, s4700, and s3377), and used at final concentration of 100 pmol per 6 well-plate for 48–72 hrs. siRNA was introduced into the cells using RNA Max Lipofectamine reagent (Invitrogen).

Techniques: Quantitative RT-PCR