Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Expression of Slc31a1 ( A ) and Slc31a2 ( B ) genes in the kidney of the suckling (14-day-old), w-t and mosaic mutant mice. The histograms display mRNA levels in arbitrary units (means ± S.D., n = 5, (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Expressing, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Co-localization of CTR1 (red channel) and aquaporin1 (AQP1, green channel), a proximal tubule marker in the kidneys of 14-day-old w-t mouse analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Bar corresponds to 20 μm.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Marker, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Immunofluorescent staining of the CTR1 protein (red, channel) analyzed by confocal microscopy in the kidney of the suckling (14-day-old). Right panels show digital enlargement of CTR1 staining visualized in left panels to reveal details of CTR1 cellular localization. This was performed using the ROI function of the ZEN programme. ( A ) Wild-type mouse, apical (arrow-head) and basolateral (long arrow) localization of CTR1 protein in the epithelial cells of the proximal renal tubules; ( B ) CuCl 2 -treated w-t mouse, visible apical (arrow-head) and cytoplasmic (little red dots (short arrow)) localization of the CTR1 protein in the epithelial cells of the proximal renal tubules; ( C ) untreated mosaic mutant, CTR1 protein exhibit apical (arrow-head) localization in the epithelial cells of the proximal renal tubules. ( D ) CuCl 2 -treated mosaic mutant, weak immunopositive signal observed in the form of the red dots, in the cytoplasm of the epithelial cells of the proximal renal tubules (short arrow) indicating for relocalization of CTR1 to the cytoplasm and degradation. Nuclei were counterstained with DAPI. Bars correspond to 20 μm.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Staining, Confocal Microscopy, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Decreased expression of the Slc31a1 ( A ) and Slc3a2 ( B ) genes in the kidney of 45-day-old copper-treated w-t and mosaic mutant mice. The histograms display mRNA levels in arbitrary units (means ± S.D., n = 5, (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Expressing, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Immunofluorescent staining of CTR1 protein (red, channel) and aquaporin 1 (AQP1), a proximal tubule marker (green channel), in the kidneys of 45-day-old w-t mouse analyzed by confocal microscopy. Localization of both CTR1 and AQP1 in the epithelial cells of the proximal renal tubules. Nuclei were counterstained with DAPI. Bars correspond to 20 μm.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Staining, Marker, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Immunofluorescent staining of CTR1 protein (red channel) analyzed by confocal microscopy in the kidney of the young (45-day-old). Right panels show digital enlargement of CTR1 staining visualized in left panels to reveal details of CTR1 cellular localization. This was performed using the ROI function of the ZEN programme: ( A ) W-t mouse, apical (arrow-head) and cytoplasmic (short arrow) localization (red dots) of CTR1 protein in the epithelial cells of the proximal renal tubules. ( B ) Cupric chloride-administered w-t mouse, cytoplasmic localization of CTR1 protein visible in the form of the red dots (short arrow) in the epithelial cells of the proximal renal tubules. ( C ) Cupric chloride-treated mosaic mutant. Cytoplasmic localization of the CTR1 protein (in the form of the red dots, short arrow) in the epithelial cells of the proximal renal tubules. Nuclei were counterstained with DAPI. Bars correspond to 20 μm.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Staining, Confocal Microscopy, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Expression of Slc31a1 ( A ) and Slc31a2 ( B ) genes in the kidney of adult (6-month-old), w-t and mosaic mutant mice. The histograms display mRNA levels in arbitrary units (means ± S.D., n = 5, (* p < 0.05, ** p < 0.01).

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Expressing, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Colocalization CTR1 (red channel) and aquaporin1 (AQP1), a proximal tubule marker (green channel) in the kidneys of adult (6-month-old) w-t mouse analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Bars correspond to 20 μm.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Marker, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Immunofluorescent staining of CTR1 protein (red, channel) analyzed by confocal microscopy in the kidney of the adult (6-month-old). Right panels show digital enlargement of CTR1 staining visualized in left panels to reveal details of CTR1 cellular localization. This was performed using the ROI function of the ZEN programme: ( A ) W-t mouse, both apical (arrow-head) and cytoplasmic (visible as numerous red dots) (short arrow) localization of CTR1 protein in the epithelial cells of the proximal renal tubules. ( B ) Cupric chloride-administered w-t mouse, apical (arrow-head) and cytoplasmic (red dots) (short arrow) localization of CTR1 protein in the epithelial cells of the proximal renal tubules. ( C ) Cupric chloride-treated mosaic mutant, strong immunopositive signal indicating for CTR1 protein expression detected in the apical membrane (arrow-head) and in the cytoplasm (red dots) of the epithelial cells of the proximal renal tubules. Expression of CTR1 protein was also observed in the cytoplasm (short arrow) of the epithelial cells of the proximal renal tubules. Nuclei were counterstained with DAPI. Bars correspond to 20 μm.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Staining, Confocal Microscopy, Mutagenesis, Expressing, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease

doi: 10.3390/ijms231911441

Figure Lengend Snippet: Schematic illustration of the CTR1 protein expression and distribution in epithelial cells of the renal proximal tubules in untreated and copper-treated w-t and mosaic mutant mice. Diagrams represent schematic structure of the proximal renal tubule, where inner and outer circles represent apical and basolateral membranes of the epithelial cells of the proximal tubule, respectively. Tubular lumen filled with yellow indicates primary urine. Membrane and/or cytoplasm localization and approximate amount of CTR1 protein (showed as red dots) was determined based on the results of the immunofluorescence analysis.

Article Snippet: The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. Non-specific antibody binding was blocked by incubating the tissue sections in PBS/3% BSA (Merck, Darmstadt, Germany) for 1.5 h. For CTR1 detection in the kidneys, the sections were incubated at RT with primary rabbit polyclonal anti-CTR1 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:100 in PBS/3% BSA.

Techniques: Expressing, Mutagenesis, Membrane, Immunofluorescence

Journal: bioRxiv

Article Title: Copper as a Novel Regulator of PERK

doi: 10.1101/2022.09.02.506340

Figure Lengend Snippet: A) Representative western blot images, probed as indicated, of WT and KO PERK (gray) MEF whole cell lysates from cells treated with 15uM TTM for 24 hours and 300nM Tg for 2hrs. B) Quantification of n=3-4 replicates of the experiments in A. One-way repeated measures ANOVA returned significant effect of treatment for WT. p<0.0001. Holm-Sidak’s multiple comparison test: *p<0.05; **p<0.01. One-way repeated measures ANOVA did not return significance for KO PERK. p=0.185. C) Representative western blot images, probed as indicated, of MEF whole cell lysates from cells treated with 15uM TTM for 24 hours and 50uM Sal for 24hrs. D) Quantification of n=3 replicates of the experiment in C. One-way repeated measures ANOVA did not return significance. p=0.13. E) Representative western blot images, probed as indicated, of WT and KO PERK (gray) MEF whole cell lysates from cells treated with 500uM BCS for 24 hours and 10uM PA for 1 hour. F) Quantification of n=4 replicates of the experiment in E. One-way repeated measures ANOVA returned significant effect of treatment for WT. p=0.0046. Holm-Sidak’s multiple comparison test: *p<0.05. One-way repeated measures ANOVA did not return significance for KO PERK. p=0.163. G) Representative western blot images, probed as indicated, of MEF whole cell lysates from cells treated with 500uM BCS for 24 hours and 50uM Sal for 24 hours. H) Quantification of n=3 replicates of the experiment in G. One-way repeated measures ANOVA did not return significance. p=0.054. I) Representative western blot images, probed as indicated, of CTR1 and KO CTR1 (gray) whole cell lysates from cells treated with 300nM Tg for 2 hours. J) Quantification of n=4 replicates of the experiment in I. Two-way repeated measures ANOVA returned treatment as a significant factor. p=0.014. Sidak’s was used for multiple comparisons. *p<0.05. K) Representative western blot images, probed as indicated, of ATP7A +/+ and ATP7A -/- (gray) whole cell lysates from cells treated with 300nM Tg for 2 hours. L) Quantification of n=4 replicates of the experiment in K. Two-way repeated measures ANOVA returned treatment as a significant factor, p<0.0001, as well as a significant interaction between treatment and genotype, p=0.023. Sidak’s test was used for multiple comparisons. **p<0.01. M) Quantification of n=3 replicates of experiment where MEFs were treated with 100uM Cu-ATSM for 1hr before being harvested and whole cell lysates immunoblotted for peIF2α, eIF2α, Actin and PERK. Two-way repeated measures ANOVA returned treatment and genotype interaction as a significant factor, p<0.05. Sidak’s test was used for multiple comparisons. *p<0.05. Tg=thapsigargin; PA=PERK Activator; Sal=Salubrinal; TTM=Tetrathiolmolybdate; BCS=Bathocuproine Disulfonic Acid; CCS=copper chaperone for SOD1; KO=knockout; MEF=mouse embryonic fibroblast.

Article Snippet: Primary antibodies and dilutions used were: 1:1000 Rabbit anit Ser 51 peIF2α (CST), 1:1000 Rabbit anti D9G8 peIF2α (CST), 1:1000 Mouse anti eIF2α (CST), 1:1000 Rabbit anti PERK (CST), 1:1000 Rabbit anti FLAG (CST), 1:40,0000 Mouse anti B-Actin (CST), 1:1000 Rabbit anti Calcineurin (CST), 1:1000 Mouse anti CHOP (CST), 1:1000 Rabbit anti Cleaved Caspase 3 (CST), 1:1000 Rabbit anti CTR1 from (CST), 1:1000 Rabbit anti CCS from (Abcam), 1:1000 Rabbit anti ATP7A (Sigma), 1:1000 Rabbit anti HRI (Sigma) and 1:2000 Mouse anti BiP (BD).

Techniques: Western Blot, Knock-Out

Journal: bioRxiv

Article Title: Copper as a Novel Regulator of PERK

doi: 10.1101/2022.09.02.506340

Figure Lengend Snippet: A) LDH Assay results quantifying LDH release as a measure of cytotoxicity from WT (black) and KO PERK (grey) MEFs treated with 300nM Tg for 20 hours. n=3. B) LDH Assay results quantifying LDH release as a measure of cytotoxicity from WT (black) and KO PERK MEFs (grey) treated with 0.5ug/mL Tm for 20 hours. n=3. C) LDH Assay results quantifying LDH release as a measure of cytotoxicity from ATP7A +/+ (black) and ATP7A -/- (grey) MEFs treated with 1uM Tg for 20 hours. n=3. D) LDH Assay results quantifying LDH release as a measure of cytotoxicity from ATP7A +/+ (black) and ATP7A -/- (grey) MEFs treated with 0.5ug/mL Tm for 20 hours. n=3. E) Representative western blot images of protein lysates from ATP7A +/+ (black) and ATP7A -/- (grey) MEFs treated with 300nM Tg or 0.5ug/mL Tm for 20 hours. n=3. F) Western blot images of protein lysates from WT (black) and KO PERK (gray) whole cell MEF lysates, treated for 20hrs with 300nM Tg or 0.5ug/mL Tm for 20 hours. n=1. G) Representative western blot images of protein lysates from CTR1 (black) and KO CTR1 (grey) MEFs treated with 300nM Tg or 0.5ug/mL Tm for 20 hours. n=3. Tg=thapsigargin; Tm=tunicamycin; Casp-3=Cleaved Caspase-3; KO=knockout; MEF=mouse embryonic fibroblast; LDH=lactose dehydrogenase; BA=Brefedlin A.

Article Snippet: Primary antibodies and dilutions used were: 1:1000 Rabbit anit Ser 51 peIF2α (CST), 1:1000 Rabbit anti D9G8 peIF2α (CST), 1:1000 Mouse anti eIF2α (CST), 1:1000 Rabbit anti PERK (CST), 1:1000 Rabbit anti FLAG (CST), 1:40,0000 Mouse anti B-Actin (CST), 1:1000 Rabbit anti Calcineurin (CST), 1:1000 Mouse anti CHOP (CST), 1:1000 Rabbit anti Cleaved Caspase 3 (CST), 1:1000 Rabbit anti CTR1 from (CST), 1:1000 Rabbit anti CCS from (Abcam), 1:1000 Rabbit anti ATP7A (Sigma), 1:1000 Rabbit anti HRI (Sigma) and 1:2000 Mouse anti BiP (BD).

Techniques: Lactate Dehydrogenase Assay, Western Blot, Knock-Out