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: iScience

Article Title: Hepatic depletion of nucleolar protein mDEF causes excessive mitochondrial copper accumulation associated with p53 and NRF1 activation

doi: 10.1016/j.isci.2023.107220

Figure Lengend Snippet:

Article Snippet: rabbit-anti-CTR1 polyclonal , ABclonal , CatA10109;RRID: AB_2757632.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Isolation, Software

Journal: Parasites & Vectors

Article Title: Role of cuproptosis in mediating the severity of experimental malaria-associated acute lung injury/acute respiratory distress syndrome

doi: 10.1186/s13071-024-06520-1

Figure Lengend Snippet: The effect of DSF or TTM on pulmonary cuproptosis-related marker expression in the experimental MA-ALI/ARDS mice. A–C Representative images of pulmonary cuproptosis-related marker (SLC31A1, ATP7A, and FDX1) expression in different groups using immunohistochemical staining: a naïve mice; b P. berghei ANKA-infected control mice at 8 dpi; c P. berghei ANKA-infected control mice at 15 dpi; d DSF-treated uninfected mice; e DSF-treated P. berghei ANKA-infected mice at 8 dpi; f DSF-treated P. berghei ANKA-infected mice at 15 dpi; g TTM-treated uninfected mice; h TTM-treated P. berghei ANKA-infected mice at 8 dpi; i TTM-treated P. berghei ANKA-infected mice at 15 dpi. D The IOD/area of positively stained cuproptosis-related marker expression was calculated from more than 20 lung fields per animal. The differences in IOD/area of positive expression between two groups or among multiple groups were compared using the independent-samples t -test or one-way ANOVA, respectively. Experiments were conducted with six mice per group, and data are presented as mean ± SD. * P < 0.05 and ** P < 0.01 vs. the P. berghei ANKA-infected control mice at 8 dpi; # P < 0.05 and ## P < 0.01 vs. the P. berghei ANKA-infected control mice at 15 dpi. NS = non-significant, P > 0.05, relative to naïve mice

Article Snippet: After routine deparaffinization, hydration, antigen retrieval, remove of endogenous peroxidase, and blockage of non-specific binding, the slices were incubated with monoclonal mouse anti-CD68 antibody (1:200; no. GB14043-50; Servicebio), polyclonal rabbit anti-SLC31A1 antibody (1:500; no. NB100-402; Novus Biologicals), or polyclonal rabbit anti-FDX1 antibody (1:200; no. 12592-1-AP; Proteintech) overnight at 4 °C, followed by incubation with secondary antibodies [Alexa Fluor ® 488 goat anti-mouse IgG (1:1000; no. 4480S; CST) and CoraLite ® 594 goat anti-rabbit IgG (H + L) (1:200; no. SA00013-4; Proteintech)] at 37 °C for 1 h in the dark.

Techniques: Marker, Expressing, Immunohistochemical staining, Staining, Infection, Control

Journal: Parasites & Vectors

Article Title: Role of cuproptosis in mediating the severity of experimental malaria-associated acute lung injury/acute respiratory distress syndrome

doi: 10.1186/s13071-024-06520-1

Figure Lengend Snippet: The effect of DSF or TTM on changes in the numbers of SLC31A1 + -CD68 + and FDX1 + -CD68 + pulmonary macrophages in experimental MA-ALI/ARDS mice. A , B Representative images of pulmonary SLC31A1 + -CD68 + and FDX1 + -CD68 + macrophages in different groups using double immunofluorescence staining under a fluorescence microscope at a magnification of ×200. The positively stained CD68 + macrophages appear green, while the positively SLC31A1 + or FDX1 + cells appear red. Co-expression of SLC31A1 + -CD68 + or FDX1 + -CD68 + cells exhibits a yellow color. C The numbers of positively SLC31A1 + -CD68 + or FDX1 + -CD68 + pulmonary macrophages were calculated from more than 20 lung fields per animal. Differences in the numbers of SLC31A1 + -CD68 + and FDX1 + -CD68 + macrophages between two groups or among multiple groups were compared using the independent-samples t -test or one-way ANOVA, respectively. Experiments were conducted with six mice per group, and data are presented as mean ± SD. * P < 0.05 and ** P < 0.01 vs. the P. berghei ANKA-infected control mice at 8 dpi; # P < 0.05 and ## P < 0.01 vs. the P. berghei ANKA-infected control mice at 15 dpi. NS = non-significant, P > 0.05, relative to naïve mice

Article Snippet: After routine deparaffinization, hydration, antigen retrieval, remove of endogenous peroxidase, and blockage of non-specific binding, the slices were incubated with monoclonal mouse anti-CD68 antibody (1:200; no. GB14043-50; Servicebio), polyclonal rabbit anti-SLC31A1 antibody (1:500; no. NB100-402; Novus Biologicals), or polyclonal rabbit anti-FDX1 antibody (1:200; no. 12592-1-AP; Proteintech) overnight at 4 °C, followed by incubation with secondary antibodies [Alexa Fluor ® 488 goat anti-mouse IgG (1:1000; no. 4480S; CST) and CoraLite ® 594 goat anti-rabbit IgG (H + L) (1:200; no. SA00013-4; Proteintech)] at 37 °C for 1 h in the dark.

Techniques: Double Immunofluorescence Staining, Fluorescence, Microscopy, Staining, Expressing, Infection, Control

Journal: Parasites & Vectors

Article Title: Role of cuproptosis in mediating the severity of experimental malaria-associated acute lung injury/acute respiratory distress syndrome

doi: 10.1186/s13071-024-06520-1

Figure Lengend Snippet: The effect of co-culture of DSF-CuCl 2 or TTM-CuCl 2 on the mRNA levels of SLC31A1, ATP7A, FDX1, CD86, CD206, TNF-α, iNOS, TGF-β, and IL-10 in iRBC-stimulated RAW 264.7 cells in vitro. The RAW 264.7 cells were pretreated with 5.0 × 10 6 iRBCs for 6 h, then treated with PBS, DSF-CuCl 2 , or TTM-CuCl 2 for 24 or 48 h. The qPCR method was used to quantify the mRNA levels of target genes. & P < 0.05 and && P < 0.01 vs. RAW 264.7 cells treated with only PBS for 24 h; * P < 0.05 and ** P < 0.01 vs. iRBC-stimulated RAW 264.7 cells for 24 h; $ P < 0.05 and $$ P < 0.01 vs. RAW 264.7 cells treated with only PBS for 48 h; # P < 0.05 and ## P < 0.01 vs. iRBC-stimulated RAW 264.7 cells for 48 h. Data are taken as mean ± SD. The experiment was conducted 4–5 times with similar results

Article Snippet: After routine deparaffinization, hydration, antigen retrieval, remove of endogenous peroxidase, and blockage of non-specific binding, the slices were incubated with monoclonal mouse anti-CD68 antibody (1:200; no. GB14043-50; Servicebio), polyclonal rabbit anti-SLC31A1 antibody (1:500; no. NB100-402; Novus Biologicals), or polyclonal rabbit anti-FDX1 antibody (1:200; no. 12592-1-AP; Proteintech) overnight at 4 °C, followed by incubation with secondary antibodies [Alexa Fluor ® 488 goat anti-mouse IgG (1:1000; no. 4480S; CST) and CoraLite ® 594 goat anti-rabbit IgG (H + L) (1:200; no. SA00013-4; Proteintech)] at 37 °C for 1 h in the dark.

Techniques: Co-Culture Assay, In Vitro

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