Journal: Experimental & Molecular Medicine

Article Title: ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses

doi: 10.1038/s12276-021-00591-1

Figure Lengend Snippet: a , b Zinc levels in the serum ( a ) and knee joint synovial fluid ( b ) of CIA mice and nonimmunized (NI) control mice ( N = 10 mice per group). c Representative images of cellular zinc levels in the joint tissues of CIA and NI mice ( N = 5 mice per group). c, cartilage; s, synovium. d Relative mRNA levels of ZIP family (zinc importer) and ZNT family (zinc exporter) members in total synovial cells were quantified by qRT-PCR analysis ( N = 6 mice per group). The error bars represent ±SD, and unpaired two-tailed Student’s t -tests were performed to determine differences between groups ( a – d ). e Representative images of immunostaining for MMP3, ZIP8, MTF1, and MT1/MT2 in the synovium of CIA and NI mice ( N = 5 mice per group). f Microarray analysis of the indicated ZIP family members in synovial tissues from patients with rheumatoid arthritis (RA; N = 33), patients with osteoarthritis (OA; N = 26), and healthy individuals (HE; N = 20). Microarray data were obtained from the Gene Expression Omnibus database at the National Center for Biotechnology Information (NCBI). The box indicates the 25th and 75th percentiles, with the centerline representing the mean; significance was analyzed by one-way ANOVA followed by Bonferroni’s post hoc comparison. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns, not significant. Scale bars: 50 μm.

Article Snippet: ZIP8 in isolated CD4 + T cells was analyzed by immunofluorescence microscopy using rabbit anti-ZIP8 antibodies (Alomone Labs).

Techniques: Quantitative RT-PCR, Two Tailed Test, Immunostaining, Microarray, Expressing

Journal: Experimental & Molecular Medicine

Article Title: ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses

doi: 10.1038/s12276-021-00591-1

Figure Lengend Snippet: a , b Typical immunofluorescence microscopic images of DAPI, ZIP8, and markers for B cells (B220), T cells (CD4), macrophages (CD11b), and fibroblast-like synoviocytes (FLSs, vimentin) in synovial tissues (left) and primary cultures of total synovial cells isolated from CIA mice (right) ( a ). The percentage of ZIP8-positive cells was determined by immunofluorescence microscopic analysis of primary cultures of total synovial cells isolated from CIA mice ( b N = 5 mice). c Flow cytometric analysis of ZIP8 expression on CD4 + cells, CD11b + cells, and B220 + cells from total synovial cells isolated from the knee joints of CIA mice. The data shown in ( a , c ) are representative of five independent experiments.

Article Snippet: ZIP8 in isolated CD4 + T cells was analyzed by immunofluorescence microscopy using rabbit anti-ZIP8 antibodies (Alomone Labs).

Techniques: Immunofluorescence, Isolation, Expressing

Journal: Experimental & Molecular Medicine

Article Title: ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses

doi: 10.1038/s12276-021-00591-1

Figure Lengend Snippet: a CD4 + T cells were isolated from WT mice (C57BL/6 and DBA/1 J) and stimulated with 5 μg/mL anti-CD3 and anti-CD28 antibodies. The mRNA and protein levels of ZIP8 were determined at the indicated time points. b Representative confocal microscopic images of ZIP8 in primary cultures of mouse CD4 + T cells stimulated with anti-CD3 and anti-CD28 antibodies for 24 h. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). c , d Representative immunoblot image ( c ) and flow cytometric analysis ( d ) of ZIP8 in isolated CD4 + Tnaive and Tem cells. e Analysis of zinc influx in isolated Tnaive and Tem cells from Slc39a8 f/f and Slc39a8 f/f ; CD4 - Cre mice. The arrow indicates the time of ZnCl 2 treatment (45 μM). The data shown in ( a – e ) are representative of three independent experiments. The values are presented as mean ± SD. * P < 0.05, ** P < 0.005.

Article Snippet: ZIP8 in isolated CD4 + T cells was analyzed by immunofluorescence microscopy using rabbit anti-ZIP8 antibodies (Alomone Labs).

Techniques: Isolation, Staining, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses

doi: 10.1038/s12276-021-00591-1

Figure Lengend Snippet: a – c Tnaive and Tem cells were isolated from WT ( Slc39a8 f/f ) and cKO ( Slc39a8 f/f ; CD4 - Cre ) mice and stimulated with or without anti-CD3 and anti-CD28 antibodies. Representative images of immunoblots used to detect the binding of CD4 to Lck ( a ), to detect phospho-TCRζ, phospho-Zap70 and Shp-1 ( b ), and to examine ERK and JNK phosphorylation and IκBα degradation ( c ). d , e The proliferation of Tnaive and Tem cells from Slc39a8 f/f and Slc39a8 f/f ; CD4 - Cre mice stimulated with anti-CD3 and anti-CD28 antibodies was measured by [ 3 H]thymidine incorporation ( d ) and CellTrace Violet (CTV) staining ( e ). The data in ( d ) were assessed with unpaired two-tailed Student’s t -tests. * P < 0.05, ** P < 0.005. f The role of ZIP8 in CD4 + T cell-mediated RA pathogenesis. Effector CD4 + T cell activation and function require ZIP8 upregulation to increase zinc influx. Thus, ZIP8 depletion in CD4 + T cells blocked CD4 + T cell-mediated RA pathogenesis by inhibiting the supply of zinc to the enlarged CD4 + T cells.

Article Snippet: ZIP8 in isolated CD4 + T cells was analyzed by immunofluorescence microscopy using rabbit anti-ZIP8 antibodies (Alomone Labs).

Techniques: Isolation, Western Blot, Binding Assay, Staining, Two Tailed Test, Activation Assay

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: (A) Representative immunoblot (top) and quantification (bottom) of ZIP8 levels in proliferating myoblasts and differentiated cells for 24, 48, and 72 h in wild type (left) and Zip8 shRNA expressing cells (right). (B) Representative immunoblot of ZIP14 levels in proliferating and differentiating wild type (left) and Zip14 shRNA myoblasts (right). For all samples, shown is the mean ± SE of three independent biological replicates. Immunoblots against actin or Coomasie-stained membranes (Supp. Fig. 8) were used as loading controls. Samples were compared to the corresponding wild type or mutant proliferation time point, and the; mutants were compared also to the equivalent time point in wild type cells. *****P<0.0001 ****P<0.001, ***P ˂ 0.005, **P ˂ 0.01

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Western Blot, shRNA, Expressing, Staining, Mutagenesis

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: (A) Representative light micrographs of wild type, Zip8 and Zip14 knockdown myoblasts grown in proliferating conditions or at 24, 48 and 72 h after inducing differentiation. Cells were immunostained with an anti-Myogenin antibody. Calculated fusion index for Zip8- (B) and Zip14-shRNA treated myoblasts. (C) Data represent mean ± SE for three independent experiments. *****P<0.0001

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Knockdown, shRNA

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: Whole cell metal content analyses of proliferating and differentiating wild type or myoblasts transduced with scrambled (scr), Zip8 or Zip14 shRNA. Statistical analyses showed significant differences in metal accumulation in differentiating myoblast comparing to the corresponding time point of wild type cells. (A) Manganese. (B) Iron. (C) Zinc. (D) Calcium. All data were obtained using ICP-OES and normalized to total protein. Shown is mean ± SE for three independent biological replicates. ****P<0.001, ***P ˂ 0.005.

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Transduction, shRNA

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: Representative Western blots, activity gels (top panels) and quantification of SOD2 levels and activity (bottom panels) in wild type and Zip8 shRNA cell (A), and Zip14 shRNA cells. For all samples, blots against actin and Coomassie-stained membranes were used as loading controls (Supp. Fig. 8). Shown is mean ± SE for three biological replicates. For wild type differentiating myoblasts, statistical analyses showed significant differences when compared to proliferating cells. Bonferroni statistical analyses for Zip8-knock down cells showed significant differences when compared to control cells at the corresponding time points, and also when compared to the mutant’s proliferation point. Similar to control cells, Zip14-knockdown cells showed significant differences when compared to proliferating Zip14-mutant myoblasts; *****P<0.0001 ****P<0.001, ***P ˂ 0.005, **P ˂ 0.01, *P ≤ 0.05

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Western Blot, Activity Assay, shRNA, Staining, Knockdown, Control, Mutagenesis

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: Cell counting assay of proliferating wild type myoblasts, myoblasts transduced with scrambled shRNA (shRNA scr, B,C), Zip8 (A) or Zip14 shRNAs (B). Data in A-C are mean ± SE for three independent experiments. *****P<0.0001 relative to 0 h of proliferation. (C) Representative light micrographs of proliferating wild type myoblasts, Zip8 or Zip14 knockdown myoblasts immunostained for Pax7.

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Cell Counting, Transduction, shRNA, Knockdown

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: (A) Representative Western blot of myoblasts transduced with the murine wild type Zip8. Detection of ZIP8, an anti-HA antibody was used to detect the tag associated to the exogenous protein. Myogenin, Caspase 3 and SOD2 antibodies were used. Actin is the loading control. SOD2 activity was also evaluated in gel. (B) Representative light micrographs of differentiating wild type myoblasts and those transduced with an shRNA against Zip8 (shRNA-2) complemented with the murine wild type Zip8 at 48 h post differentiation. Cells were immunostained with an anti-Myogenin antibody. (C) Cell counting assay of proliferating wild type myoblasts, cells transduced with scrambled shRNA (scr shRNA), and cells complemented with Zip8. Data is the mean ± SE for three independent experiments. ICP analysis of (D) Mn, (E) Fe, (F) Zn in cells Zip8 shRNA cells complemented with wild type Zip8.

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Western Blot, Transduction, Control, Activity Assay, shRNA, Cell Counting

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: (A) Representative Western blot of myoblasts transduced with the murine wild type Zip8 and Zip14. The antibodies used were ZIP8, ZIP14, Myogenin, Caspase 3 and SOD2 antibodies were used. Actin is the loading control. SOD2 activity was also evaluated in gel. (B) Representative light micrographs of differentiating wild type myoblasts and those transduced with scramble and with both shRNA against Zip8 and Zip14 at 24 h post differentiation. Cells were immunostained with an anti-Myogenin antibody. (C) Cell counting assay of proliferating wild type myoblasts, scrambled shRNA and double mutant. Data is the mean ± SE for three independent experiments. Whole cell metal content analyses of proliferating and differentiating wild type or myoblasts transduced with scrambled (scr), Zip8 and Zip14 shRNA. Statistical analyses showed significant differences in metal accumulation in differentiating myoblasts (D) Manganese. (E) Iron. (F) Zinc. Data was obtained using ICP-OES and normalized to total protein. Shown is mean ± SE for three independent biological replicates. ****P<0.001, ***P ˂ 0.005

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Western Blot, Transduction, Control, Activity Assay, shRNA, Cell Counting, Mutagenesis

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: Cell counting assays performed in myoblasts expressing Zip8 and Zip14 shRNAs grown in medium supplemented with exogenous Mn (A), Fe (B) or Zn (C). Statistical analyses showed significant differences when comparing differentiating myoblasts to proliferating cells. For all experiments, data represent mean ± standard error for three biological replicates. *****P<0.0001. (D) Representative light micrographs of differentiating wild type myoblasts and those transduced with scramble and shRNA against Zip8 or Zip14 at 48 h post differentiation. Cells were immunostained with an anti-Myogenin antibody.

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: Cell Counting, Expressing, Transduction, shRNA

Journal: Metallomics : integrated biometal science

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1039/c8mt00348c

Figure Lengend Snippet: Mn, Fe and Zn levels in proliferating and differentiating cells supplemented with either metal.

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340), anti-Caspase 3 (A2156), anti-HA (AE008) from Abclonal.

Techniques: shRNA

Journal: bioRxiv

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1101/494542

Figure Lengend Snippet: Whole cell metal content analyses of proliferating and differentiating wild type or myoblasts transduced with scrambled (scr), Zip8 or Zip14 shRNA. Statistical analyses showed significant differences in metal accumulation in differentiating myoblasts ( A ) Manganese. ( B ) Iron. (C) Zinc. ( D ) Calcium. All data were obtained using ICP-OES and normalized to total protein. Shown is mean ± standard error for three independent biological replicates. ****P<0.001, ***P < 0.005

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340) anti-Caspase 3 (A2156) from Abclonal.

Techniques: Transduction, shRNA

Journal: bioRxiv

Article Title: Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

doi: 10.1101/494542

Figure Lengend Snippet: (A-C) Cell counting assay of proliferating wild type myoblasts, myoblasts transduced with scrambled shRNA (shRNA scr, B,C), Zip8 ( B ) or Zip14 shRNAs ( C ). Data in A-C are mean ± standard error for three independent experiments. *****P<0.0001. (D-F) Representative light micrographs of proliferating wild type myoblasts ( D ), Zip8 ( E ) or Zip14 ( F ) knockdown myoblasts immunostained for Pax7.

Article Snippet: The proteins of interest were detected using the primary antibodies rabbit anti-ZIP8 (A10395), anti-ZIP14 (A1043), anti-SOD1 (A0274), anti-SOD2 (A1340) anti-Caspase 3 (A2156) from Abclonal.

Techniques: Cell Counting, Transduction, shRNA

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Müller Cell Ablation Leads to Retinal Iron Accumulation

doi: 10.1167/iovs.17-21743

Figure Lengend Snippet: Iron accumulation in the primary MCA model leads to differential regulation of the NTBI importers Zip8 and Zip14. There is no change in Zip8 mRNA levels in the MCA and control retinas at 2 weeks (A) or 5 months (E) post ablation. There is no change in Zip8 protein levels between the MCA and control retinas at 2 weeks post ablation (C), but there is a significant increase in Zip8 protein levels in the MCA retinas compared with the control retinas at 5 months post ablation (G). There is a significant decrease in Zip14 mRNA levels between the MCA and control retinas at 2 weeks post ablation (B) but no change in Zip14 mRNA levels at 5 months post ablation (F). There is a significant decrease in Zip14 protein levels in the MCA retinas compared with controls at 2 weeks post ablation (D), but no change at 5 months post ablation (H). Loading control β-Actin (42kDa) bands are shown below each set of lanes. *P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies used were as follows: rat anti-transferrin receptor (Serotec), rabbit anti-Dmt1 (NRAMP24-A; Alpha Diagnostic International, San Antonio, TX, USA), rabbit anti-Zip8 (ThermoScientific, Philadelphia, PA, USA), rabbit anti-Zip14 (ThermoScientific), rabbit anti-Fpn (Pierce, Chicago, IL, USA), rabbit anti-ferritin (E17) (P. Arosio), and mouse anti-beta actin (ab8226; Abcam).

Techniques: