Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a qPCR analysis of Slc39a8 expression in 8-week-old C57BL/6J female (left) and male (right) mouse tissues ( n = 6 per group). Small intestines were equally divided into 7 segments, and each segment is approximately 6 cm in length (Sm. int.1–7). “Small intestine 1” refers to duodenum; “Small intestine 4” refers to jejunum; “Small intestine 7” refers to ileum; and “Large intestine” refers to distal colon. b Fluorescence images of frozen duodenal, jejunal, ileal, and colon sections from 8-week-old C57BL/6J female (upper) and male (lower) mice. DAPI (blue); SLC39A8 (red). Original magnification, ×10 and ×60 (enlarged insets); Scale bars: 100 μm. Each image was acquired independently three times, with similar results. c qPCR analysis of Slc39a8 expression in 10-week-old male control and Slc39a8- IEC KO mice ( n = 4 per group). “Duodenum” refers to the proximal 6 cm of small intestines; “Jejunum” refers to the medial 18–24 cm of small intestines; “Ileum” refers to the medial 36–42 cm of small intestines; and “Colon” refers to the distal 4–8 cm of large intestines. d ICP-MS analysis of Mn levels in duodenum, jejunum, ileum, colon, liver, bile, lung, kidney, heart, brain, whole blood from 20-week-old male (M) and female (F) control (Cont) and Slc39a8- IEC KO (KO) mice ( n = 5 male per group; n = 6 female per group). Data are presented as individual values and represent the mean ± SEM. The p -values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test for c , and unpaired two-tailed Student’s t test for d . ns not significant. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Expressing, Fluorescence, Control, Two Tailed Test

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a Control and Slc39a8- IEC KO mice at 10 weeks of age were administered 0.1 µCi [ 54 Mn]MnCl 2 per gram body weight via oral-gastric gavage. Blood was collected at 15 min, and blood counts per min (cpm) were determined by γ-counting. Mice were killed 4 h later, and ( b ) duodenum, jejunum, ileum, and colon, and ( c ) tissue cpm were determined by γ-counting. d Control and Slc39a8- IEC KO mice at 10 weeks of age were administered 0.1 µCi [ 54 Mn]MnCl 2 per gram body weight via tail vein injection. Blood was collected at 15 min, and blood counts per minute (cpm) were determined by γ-counting. Mice were killed 4 h later, and ( e ) duodenum, jejunum, ileum, and colon, and ( f ) tissue cpm were determined by γ-counting. [control: n = 4, n = 2 male (M), n = 2 female (F); Slc39a8- IEC KO: n = 4 per group, n = 2 M, n = 2 F]. Data are presented as individual values and represent the mean ± SEM. The p -values were determined by unpaired two-tailed Student’s t test for a – f . ns not significant. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Control, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a Schematic illustrating the generation of intestinal organoid monolayer cultures from murine intestinal crypts. b Representative confocal images showing immunostaining for SLC39A8 (red), ZO1 (green), and DAPI (blue) in enteroid or colonoid monolayers from control and Slc39a8- IEC KO mice. Polarized apical surfaces were stained with ZO1 (green), indicated by black arrowheads in the top panels. XY projection (bottom panels); XZ projection (top panels) and YZ projection (right panels) orthogonal views. Scale bar: 25 µm. Each image was acquired independently three times, with similar results. c Schematic representation of apical 54 Mn accumulation by intestinal organoid monolayer culture. d 54 Mn was added to the media in the apical compartments of enteroid or colonoid monolayers. Cell-associated radioactivity was determined with a gamma counter ( n = 3 biologically independent samples). e Schematic representation of basolateral 54 Mn accumulation by primary IECs. f 54 Mn was added to the media in the basolateral compartments of enteroid or colonoid monolayers. Cell-associated radioactivity was determined with a gamma counter ( n = 3 biologically independent samples). Data are presented as individual values and represent the mean ± SEM. The p -values were determined by unpaired two-tailed Student’s t test for d and f . ns not significant. Source data are provided as a Source Data file. Figures a , c , and e were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Immunostaining, Control, Staining, Radioactivity, Two Tailed Test

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a Schematic of the DSS-induced acute colitis model. For colitis induction, mice were given drinking water containing 3% (w/v) DSS for 8 days (inflammatory phase). The mice were then provided with regular drinking water for 5 days (recovery phase). Representative data from two independent experiments are shown. b Changes in body weight (percentage of original body weight) over time (days) in 8-week-old male control and Slc39a8- IEC KO mice following DSS treatment ( n = 13 per group). c Gross morphology of the colon. d Colon length and e spleen/body weight ratios on day 13 after the DSS treatment in control and Slc39a8- IEC KO mice ( n = 6 per group). f Hematoxylin/eosin (H&E) staining of colons and g pathological scores on day 13 without or with DSS treatment in control and Slc39a8- IEC KO mice ( n = 8 per group). Scale bars: 100 μm. h Measurement of 4 kDa FITC-dextran in serum on day 13 without or with the DSS treatment in control and Slc39a8- IEC KO mice ( n = 6 per group). i qPCR quantification (control, n = 5; Slc39a8- IEC KO, n = 5; control + DSS, n = 6; Slc39a8- IEC KO + DSS, n = 6), j immunofluorescence staining and relative fluorescence, and k immunoblot analysis and densitometric quantification of tight junction proteins in the colons of control and Slc39a8- IEC KO mice on day 13 without or with DSS treatment ( n = 3 per group). Scale bar: 100 μm. l qPCR quantification of proinflammatory cytokines and chemokines in the colons of control and Slc39a8- IEC KO mice on day 13 without or with DSS treatment ( n = 5 per group). Data are presented as individual values and represent the mean ± SEM. The p -values were determined by two-way ANOVA with Bonferroni’s multiple comparisons test for b , unpaired two-tailed Student’s t test for d and e , and one-way ANOVA with Bonferroni’s multiple comparisons test for g – l . Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Control, Staining, Immunofluorescence, Fluorescence, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a Schematic of the DSS-induced chronic colitis model. For colitis induction, mice received two cycles of DSS treatment provided in drinking water. Each cycle consisted of 8 days of water containing 3% DSS (inflammatory phase) followed by 5 days of water alone (recovery phase). Representative data from two independent experiments are shown. b Time course of the disease activity index in control and Slc39a8- IEC KO mice during the second DSS cycle ( n = 6 per group). c Colon length from control and Slc39a8- IEC KO mice on day 14 without or with DSS treatment (control, n = 5; Slc39a8- IEC KO, n = 5; control + DSS, n = 6; Slc39a8- IEC KO + DSS, n = 6 per group). d Survival percentage (%) over time. e H&E staining of colons and f pathological scores from control and Slc39a8- IEC KO mice on day 14 without or with DSS treatment (control, n = 5; Slc39a8- IEC KO, n = 5; control + DSS, n = 6; Slc39a8- IEC KO + DSS, n = 6 per group). Scale bars: 100 μm. Data are presented as individual values and represent the mean ± SEM. The p -values were determined by two-way ANOVA with Bonferroni’s multiple comparisons test for b , one-way ANOVA with Bonferroni’s multiple comparisons test for c and f , and log-rank test for d . Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Activity Assay, Control, Staining

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a RNA-seq analysis revealed 4 misregulated genes ( P adj < 0.1, n = 3) in the intestines of 8-week-old male Slc39a8- IEC KO mice. Volcano plot profiles of the -log10 adjusted p -value and log2 fold change of gene expression between control and Slc39a8- IEC KO mice intestines. b Genome browser shot of the Acer1 locus. Validation of Acer1 expression by qPCR in c the ileal and colonic mucosa in control and Slc39a8- IEC KO mice ( n = 3 per group), and d the enteroid and colonoid monolayers derived from control or Slc39a8- IEC KO mice ( n = 4 biologically independent samples per group). Validation of Acer1 protein expression by immunoblot analysis in e the ileal and colonic mucosa in control and Slc39a8- IEC KO mice ( n = 3 per group), and f the enteroid and colonoid monolayers derived from control and Slc39a8 -IEC KO mice ( n = 2 biologically independent samples). Immunoblots of actin were used as the loading control. Quantification of the relative protein expression after normalization with actin. Data are presented as individual values and represent the mean ± SEM. The p -values were determined by Wald test implemented in DESeq2 R package for a , and unpaired two-tailed Student’s t test for c – e . Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: RNA Sequencing, Gene Expression, Control, Biomarker Discovery, Expressing, Derivative Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: qPCR analysis of tight junction ( a ) Zo1 , ( b ) Cldn3 , ( c ) Cldn5 , and ( d ) Cldn7 measured 24 h after D- e -MAPP treatment in Slc39a8- IEC KO mouse-derived enteroid or colonoid monolayers (Without D- e -MAPP treatment, n = 4; D- e -MAPP treatment, n = 3 biologically independent samples). e Immunoblot analysis and densitometric quantification of ZO1, Cldn3, Cldn5, and Cldn7 measured 24 h after D- e -MAPP treatment in Slc39a8- IEC KO mouse-derived enteroid monolayers. Quantification of the relative protein expression after normalization with actin ( n = 2 biologically independent samples). f Paracellular flux of 4 kDa FITC-dextran and g Intestinal transepithelial electrical resistance (TEER) were measured 24 h after D- e -MAPP treatment in Slc39a8- IEC KO mouse-derived enteroid and colonoid monolayers (Without D- e -MAPP treatment, n = 4; D- e -MAPP treatment, n = 3 biologically independent samples). Data are presented as individual values and represent the mean ± SEM. The p -values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test for a – d , f , and g . Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Derivative Assay, Western Blot, Expressing

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a Schematic of pretreatment with the ACER1 inhibitor D- e -MAPP in the DSS-induced colitis model in mice. Mice were treated with or without the ACER1 inhibitor D- e -MAPP (10 nmol/g body weight) daily for one week, followed by DSS treatment to induce colitis. b Changes in body weight (percentage of original body weight) over time (days) in 8-week-old male mice following DSS treatment ( n = 4 per group). c Gross morphology of the large intestine. d Colon length on day 14 after the DSS treatment ( n = 4 per group). e Hematoxylin/eosin (H&E) staining of colons and f pathological scores on day 14 after DSS treatment ( n = 4 per group). Scale bars: 100 μm. g Measurements of 4-kDa FITC-dextran in serum on day 14 after the DSS treatment in control and Slc39a8- IEC KO mice ( n = 4 per group). qPCR quantification of ( h ) tight junction and ( i ) proinflammatory cytokines and chemokines in colon mucosa on day 13 after DSS treatment in control and Slc39a8- IEC KO mice ( n = 4 per group). j Sphingolipid metabolism. k Heat map showing significantly altered specific lipids in the intestines of control and Slc39a8 -IEC KO mice with and without treatment with the ACER1 inhibitor D- e -MAPP. Shades of red and green represent upregulated and downregulated lipids, respectively (see color key). Lipid classes include SM sphingomyelins, HexCer hexosylceramides, SPB sphinganine, PE-Cer phosphatidylethanolamine ceramides, and CE: ceramides. n = 3 per group. l SMS1 activity in control and Slc39a8 -IEC KO intestines ( n = 6 per group) and intestinal organoid monolayer cells derived from control and Slc39a8 -IEC KO mice ( n = 5 biologically independent samples). m Average levels of three sphingomyelin species (SM 40:0;3O, SM 30:0;2O, and SM 43:1;2O). Data are presented as individual values and represent the mean ± SEM. The p -values were determined by two-way ANOVA with Bonferroni’s multiple comparisons test for b , one-way ANOVA with Bonferroni’s multiple comparisons test for d , f – i , and unpaired two-tailed Student’s t test for l . Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Staining, Control, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

doi: 10.1038/s41467-024-49049-8

Figure Lengend Snippet: a Schematic of the dietary Mn restriction experiment and treatment with the ACER1 inhibitor D- e -MAPP in the DSS-induced mouse model of colitis. Mice were fed either Mn-deficient (Mn-D) or Mn-adequate (Mn-A) diets for 14 days. Mice were then treated with or without D- e -MAPP (10 nmol/g body weight) daily at 24 h intervals during the DSS treatment (inflammatory phase). The mice were then provided with regular drinking water (recovery phase). b ACER1 expression in the ileal and colonic mucosa in Mn-A and Mn-D mice ( n = 6 per group). c Changes in body weight (percentage of original body weight) over time (days) in Mn-A and Mn-D mice following DSS treatment ( n = 15 per group). d Survival percentage (%) over time. e Gross morphology of the large intestine. f Colon length on day 14 after DSS treatment (Mn-A, n = 8; Mn-A + D-MAPP, n = 8; Mn-D, n = 6; Mn-D + D-MAPP, n = 8 per group). g Hematoxylin/eosin (H&E) staining of colons and h pathological scores on day 14 after DSS treatment (Mn-A, n = 8; Mn-A + D-MAPP, n = 8; Mn-D, n = 4; Mn-D + D-MAPP, n = 8 per group). Scale bars: 100 μm. i A model of the proposed mechanism by which Slc39a8 deficiency contributes to the pathophysiology of IBD. The proposed scheme shows that Slc39a8 deficiency impairs intestinal Mn absorption and disrupts the epithelial barrier by the upregulation of Acer1, thereby leading to inflammation. Data are presented as individual values and represent the mean ± SEM. The p -values were determined by unpaired two-tailed Student’s t test for b , two-way ANOVA with Bonferroni’s multiple comparisons test for c , Log-rank test for d , and one-way ANOVA with Bonferroni’s multiple comparisons test for f and h . Source data are provided as a Source Data file. Figure 9i was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50).

Techniques: Expressing, Staining, Two Tailed Test

Journal: Metallomics

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: Fig. 4 Knockdown of Zip8 impairs differentiation of primary myoblasts. (A) Representative light micrographs of wild type, Zip8 and Zip14 knock- down 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 experi- ments. *****P o 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

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: Fig. 3 ZIP8 and ZIP14 expression in proliferating and differentiating wild type and shRNA knockdown primary myoblasts derived from mouse satellite cells. (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 (Fig. S8, ESI†) 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 o 0.0001 ****P o 0.001, ***P o 0.005, **P o 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: Expressing, shRNA, Knockdown, Derivative Assay, Western Blot, Staining, Mutagenesis

Journal: Metallomics

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: Fig. 5 Knockdown of Zip8, but not Zip14 impairs metal intake in differentiating primary myoblasts. 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 o 0.001, ***P o 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: Knockdown, Transduction, shRNA

Journal: Metallomics

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: Fig. 6 SOD2 expression and activity is decreased in Zip8, but not Zip14 knockdown primary myoblasts. Representative Western blots, activity gels (top panels) and quantification of SOD2 levels and activity (bottom panels) in wild type and Zip8 shRNA cells (A), and Zip14 shRNA cells (B). For all samples, blots against actin and Coomassie-stained membranes were used as loading controls (Fig. S8, ESI†). 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 o 0.0001 ****P o 0.001, ***P o 0.005, **P o 0.01, *P r 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: Expressing, Activity Assay, Knockdown, Western Blot, shRNA, Staining, Control, Mutagenesis

Journal: Metallomics

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: Fig. 7 Partial depletion of Zip8, but not Zip14, impairs growth of primary myoblasts. (A) Cell counting assay of proliferating wild type myoblasts, myoblasts transduced with scrambled shRNA (shRNA scr), Zip8 or Zip14 shRNAs. Data are the mean SE for three independent experiments. *****P r 0.0001 relative to 0 h of proliferation. (B) Representative light micrographs of proliferating wild type myoblasts, Zip8 or Zip14 knock- down 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

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: Fig. 8 Exogenous ZIP8 rescued the proliferation and differentiation phenotype of Zip8 knockdown myoblasts. (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: Knockdown, Western Blot, Transduction, Control, Activity Assay, shRNA, Cell Counting

Journal: Metallomics

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: Fig. 9 Zip8 and Zip14 double knockdown myoblasts present severe proliferation and differentiation defects. (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 anti- bodies 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 immunos- tained with an anti-myogenin antibody. (C) Cell counting assay of pro- liferating 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 o 0.001, ***P o 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: Knockdown, Western Blot, Transduction, Control, Activity Assay, shRNA, Cell Counting, Mutagenesis

Journal: Metallomics

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: Fig. 10 Manganese supplementation partially rescues the proliferation defect, but not the differentiation phenotype of Zip8 knockdown myoblasts. 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 o 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: Knockdown, Cell Counting, Expressing, Transduction, shRNA

Journal: Clinical and Experimental Medicine

Article Title: Unveiling the prognostic and therapeutic landscape of the zinc transporter protein SLC39A family in colorectal cancer through multi-omics and machine learning approaches

doi: 10.1007/s10238-025-01996-2

Figure Lengend Snippet: SLC39A8 and SLC39A14 identified as key model genes playing important roles in tumor microenvironment and cancer progression in CRC (A-B) Representative images of SLC39A8 (A) and SLC39A14 (B) IHC staining in CRC tumors and their paired normal tissues. (C-D) GO and KEGG terms enriched for DEGs between SLC39A8 (C) high/low expressing groups and SLC39A14 (D) high/low expressing groups were identified through GSEA analysis. (E-F) Differences in MsigDB-based pathway activities between SLC39A8 (E) high/low expressing groups and SLC39A14 (F) scored by GSVA. Statistical significance was calculated using Wilcoxon test (E-F). *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: Primary antibodies for SLC39A8 (Cell Signaling Technology, 47188S) and SLC39A14 (Cell Signaling Technology, 24161T) were applied overnight at 4 °C in a humidified chamber.

Techniques: Immunohistochemistry, Expressing

Journal: Annals of Translational Medicine

Article Title: MicroRNA-25-3p therapy for intervertebral disc degeneration by targeting the IL-1β/ZIP8/MTF1 signaling pathway with a novel thermo-responsive vector

doi: 10.21037/atm-20-6595

Figure Lengend Snippet: Different expression profiles of IL-1β, ZIP8, MTF1 and miRNA-25-3p between normal and degenerated nucleus pulposus (NP) tissue. (A) Representative hematoxylin-eosin (HE) and safranin-O-fast green (SO-FG) staining of normal and degenerated human NP tissue. The bar is 500 µm. (B) Relative expression levels of IL-1β mRNA, ZIP8 mRNA and MTF1 mRNA were assessed in normal samples (NS) and degenerated samples (DS) of NP tissue (n=10) via qRT-PCR. (C) Relative expression profiles of IL-1β, ZIP8 and MTF1 proteins were examined in normal and degenerated NP tissue (n=10) via western blotting. (D) Expression of miRNA-25-3p in normal and degenerated NP tissue (n=10). Symbols represent individual disc samples; bars show the mean and 95% confidence interval for each group. *, P<0.05.

Article Snippet: Membranes were incubated overnight at 4 °C with anti-IL-1β antibody (12703, 1:1,000, CST, USA), anti-MTF1 antibody (ab184119, 1:1,000, Abcam, Cambridge, UK), anti-ZIP8 antibody (NBP1-76505, 1:2,000, Novus, Biologicals, Colorado, USA), anti-MMP3 antibody (NB100-91878, 1:1,000, Novus Biologicals, Colorado, USA), anti-MMP13 antibody (NBP2-45887, 1:1,000, Novus, Biologicals, Colorado, USA), anti-ADAMTS5 antibody (NBP2-15286, 1:1,000, Novus, Biologicals, Colorado,USA), anti-β-actin antibody (abs118937, 1:3,000, Absin, Shanghai, China), and anti-PCNA antibody (abs13075, 1:2,000, Absin, Shanghai, China).

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

Journal: Annals of Translational Medicine

Article Title: MicroRNA-25-3p therapy for intervertebral disc degeneration by targeting the IL-1β/ZIP8/MTF1 signaling pathway with a novel thermo-responsive vector

doi: 10.21037/atm-20-6595

Figure Lengend Snippet: Dose-dependent changes in ZIP8 and MTF1 protein expression after IL-1β stimulation in nucleus pulposus (NP) cells. Human NP cells were cultured for 72 hours in serum with different concentrations of IL-1β. (A) ZIP8 protein expression changes with β-actin normalization. (B) Nuclear MTF1 protein expression changes with PCNA normalization. Data are the mean ± standard deviation (SD) of triplicate results. **, P<0.01.

Article Snippet: Membranes were incubated overnight at 4 °C with anti-IL-1β antibody (12703, 1:1,000, CST, USA), anti-MTF1 antibody (ab184119, 1:1,000, Abcam, Cambridge, UK), anti-ZIP8 antibody (NBP1-76505, 1:2,000, Novus, Biologicals, Colorado, USA), anti-MMP3 antibody (NB100-91878, 1:1,000, Novus Biologicals, Colorado, USA), anti-MMP13 antibody (NBP2-45887, 1:1,000, Novus, Biologicals, Colorado, USA), anti-ADAMTS5 antibody (NBP2-15286, 1:1,000, Novus, Biologicals, Colorado,USA), anti-β-actin antibody (abs118937, 1:3,000, Absin, Shanghai, China), and anti-PCNA antibody (abs13075, 1:2,000, Absin, Shanghai, China).

Techniques: Expressing, Cell Culture, Standard Deviation

Journal: Annals of Translational Medicine

Article Title: MicroRNA-25-3p therapy for intervertebral disc degeneration by targeting the IL-1β/ZIP8/MTF1 signaling pathway with a novel thermo-responsive vector

doi: 10.21037/atm-20-6595

Figure Lengend Snippet: MTF1 is a target gene of miRNA-25-3p. (A) Schematic representation of the MTF 3'UTR showing the putative miRNA target site. (B) Luciferase activity of the MTF1 3'UTR reporter was analyzed in nucleus pulposus (NP) cells. An miRNA-25-3p mimic and its negative control (NC) were co-transfected with the wild-type MTF1 3'UTR or mutant vector. (C) The relative MTF1 mRNA and (D) protein expression of transfected NP cells with the miRNA-25-3p mimic and the NC were assessed using qRT-PCR and western blotting, respectively. β-actin was used as an internal control. (E) Illustration depicting the IL-1β/ZIP8/MTF1 signaling pathway regulatory mechanisms and miRNA-25-3p targets. Data are represented as the mean ± standard deviation (SD). *, P<0.05.

Article Snippet: Membranes were incubated overnight at 4 °C with anti-IL-1β antibody (12703, 1:1,000, CST, USA), anti-MTF1 antibody (ab184119, 1:1,000, Abcam, Cambridge, UK), anti-ZIP8 antibody (NBP1-76505, 1:2,000, Novus, Biologicals, Colorado, USA), anti-MMP3 antibody (NB100-91878, 1:1,000, Novus Biologicals, Colorado, USA), anti-MMP13 antibody (NBP2-45887, 1:1,000, Novus, Biologicals, Colorado, USA), anti-ADAMTS5 antibody (NBP2-15286, 1:1,000, Novus, Biologicals, Colorado,USA), anti-β-actin antibody (abs118937, 1:3,000, Absin, Shanghai, China), and anti-PCNA antibody (abs13075, 1:2,000, Absin, Shanghai, China).

Techniques: Luciferase, Activity Assay, Negative Control, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Standard Deviation

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: Schematic Work-Flow Model. Starting from the top left and working across and down, scRNA-seq analysis was conducted on lung tissue obtained from WT and Zip8 KO mouse lung tissue before and after infection with S.pneumoniae . Macrophage-specific clusters were identified and then subject to genome-scale metabolic modeling via the ftINIT pipeline. These models were analyzed to identify metabolic capacities and uncover significant metabolic vulnerabilities. This revealed thatZIP8 loss was strongly associated with mitochondrial dysfunction which is associated with disruption of multiple essential metabolic processes, all of which have the potential to impair macrophage-mediated bacterial clearance.

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: Infection, Disruption

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: ( a ) Scatterplot for pathway net flux distribution in WT and Zip8 KO macrophage clusters both pre-and post infection show distinct pattern shifts. Variance magnitude was determined by calculating the fold-change of each comparison relative to the smallest observed variance (** FC >= 1, *** FC >= 1.1, and **** FC>= 1.3). ( b & c ) Heatmap and metabolic map of metabolic pathways that vary the most across all conditions-highlighting major shifts originating from all parts of metabolism.

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: Infection, Comparison

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: ( a ) Comparison of amino acid utilization in WT and Zip8 KO macrophage clusters before and after infection. Significant utilization shift was measured by fold­change of fluxes (unit: mmol/gDW/hr) **FC >= 0.5, *** FC>=l, **** FC>= 5. ( b ) Heatmap showing the Flux Sum of amino acids across three organelle compartments (cytosol, mitochondria, and lysosome). The shift in amino acid utilization and in silico concentration indicates major metabolic shifts occurring in Zip8 KO macrophage clusters.

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: Comparison, Infection, In Silico, Concentration Assay

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: ( a ) Box plot comparison of ATP production and demand in WT and Zip8 KO macrophage clusters before and after infection. ( b, c ) Comparison of differences in the predicted pool size of reactive species and oxygen in the cytoplasm and mitochondria of WT and Zip8 KO macrophage clusters. ( d ) Changes in net glycolytic flux (mmol/gDW/hr) in WT versus Zip8 KO macrophage clusters. ( e ) Schematic diagram connecting the potential for metabolic changes to influence immune function. Significant change influxes is calculated by Fold Change (FC) where ** FC >= 0.5, *** FC >= 3, **** FC >= 5.

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: Comparison, Infection

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: WT and Zip8 KO BMDMs were analyzed before and after S.pneumoniae infection for: (a) Intracellular ROS as determined by MitoSox staining; (b-g) the SeaHorse XF Mito Stress test; and (h-k) the XF Glycolysis Stress Test. As predicted by in silico analysis, Zip8 KO BDMSs have significantly increased ROS production, oxygen demand, extracellular acidification, and glycolytic activity compared to WT BMDMs that is further exacerbated in response to infection. (*p < 0.05; **p < 0.01; ***p <0.001. Data representative of a minimum of 3 independent experiments; Data were analyzed using GraphPad Prism version 10.5.0. Statistical significance was determined using one-way ANOVA followed by Sidak’s corrections for multiple comparisons between groups. Values are expressed as means + SEM).

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: Infection, Staining, In Silico, Activity Assay

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: ( a ) Heatmap of leading in silico -host-derived metabolites via Flux shift analysis reveals kynureic acid, succinate, n-Acetyl-Cysteine, and 4-aminobutyrate as lead candidates. Pretreatment of WT and Zip8 KO BMDMs with (b) kynureic acid and (c) succinate restored bacterial clearance in Zip8 KO BMDMs similar to WT controls. Pretreatment with (d) N-acetyl-cysteine and (e) 4-aminobutγrate had no beneficial effect. (* p < 0.05; ** p <0.01; Data representative of a minimum of 3 independent experiments; Data were analyzed using GraphPad Prism version 10.5.0. Statistical significance was determined using an unpaired t-test. Values are expressed as means ± SEM).

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: In Silico, Derivative Assay

Journal: bioRxiv

Article Title: A Closed Computational–Experimental Loop Identifies Metabolic Collapse at the Root of Macrophage Dysfunction due to Zinc Dyshomeostasis

doi: 10.64898/2026.01.09.698698

Figure Lengend Snippet: Based on in silico analysis, derangement of multiple metabolomic pathways appeared in Zip8 KO macrophage clusters compared to WT clusters before and after infection. Many pathways converged directly or indirectly upon the mTOR signaling pathway, a pathway that is vital for proper phagolysomal-mediated removal of bacteria. Red denotes molecules within the Zip8 KO model whose activity is predicted to be significantly decreased compared to WT models. Green denotes molecules with increased activity.

Article Snippet: To create the conditional Zip8 KO, Zip8 flox/flox mice were bred to LysMcre (The Jackson Laboratory), which is specific to myeloid cells.

Techniques: In Silico, Infection, Bacteria, Activity Assay

Journal: Sleep

Article Title: A potential role for zinc in restless legs syndrome

doi: 10.1093/sleep/zsaa236

Figure Lengend Snippet: Quantification of zinc transporter protein levels in RLS and Control peripheral lymphocytes. The levels of zinc transporter (ZIP8, ZIP14, and TRPM7) were determined with ELISA assays. No significant difference was observed in the protein levels of three zinc transporters. (A) ZIP8 (RLS: n=17, Control: 18); (B) ZIP14 (RLS: n = 15, Control: 17); (C) TPRM7 (RLS: n = 15, Control: 17). The box edges denote the upper and lower quartile range, with the box itself denoting the interquartile range, whiskers denote the full data range, and the horizontal line denotes the median data value for each group. The data were analyzed by Student’s t-test. For each experiment, n > 15 samples measured, and each sample was run in duplicate.

Article Snippet: Quantification of zinc transporter (ZIP14, ZIP8, and TRPM7) was performed using ELISA assays against human ZIP8 (MBS9317558), ZIP14 (MBS9319878), and TRPM7 (MBS9322665) from MyBiosource (San Diego, CA).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: ZIP8 KO mice have decreased liver manganese and increased splenic iron levels. ICP-MS was performed on liver and spleens from 16-week-old ZIP8 KO mice and littermate controls (fl/fl). n = 5 mice per group. (A,C) Concentrations and (B,D) total levels of (A-B) liver and (C-D) splenic iron, manganese, and zinc were measured. Graphs depict mean and SD; P values determined by 2-tailed unpaired Student t test. SD, standard deviation.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: Baseline ZIP8 KO mice have impaired iron recycling. (A-D) Tissues and serum from 16-week-old ZIP8 KO and littermate controls (fl/fl) were harvested for analysis. n = 4 to 14 mice per group. (A) Liver, spleen and serum nonheme iron levels. (B) CBCs. (C) Liver Hamp mRNA and BM Erfe mRNA levels. (D) Representative images of Perls’ staining for iron in ZIP8 KO and fl/fl FFPE spleen tissue. 40× original magnification. (E) Splenic red pulp macrophages from C57BL/6 mice were isolated via magnetic bead selection for F4/80+ cells. ZIP8 protein levels are enriched in the splenic F4/80+ population as determined by western blot. n = 4 mice per group. (A-C) Graphs depict mean and SD; P values determined by 2-tailed unpaired Student t test. BM, bone marrow; FFPE, formalin-fixed paraffin-embedded; SD, standard deviation.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Staining, Isolation, Selection, Western Blot, Formalin-fixed Paraffin-Embedded, Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: ZIP8 KO and WT mice have similar responses to PHZ. (A-C) C57BL/6 mice were treated with 300 μL of aged RBCs, a single IP injection of 10 mg iron dextran or 2 consecutive daily doses of 60 mg/kg PHZ to induce hemolytic anemia. n = 3 to 5 mice for each treatment group. Tissues were harvested 3 days after treatment. n = 3 to 5 mice per group. (A) Western blot of splenic ZIP8. (B) Correlation between splenic ZIP8 protein levels as quantified by densitometry and splenic nonheme iron levels. P value and R 2 as determined by Pearson’s correlation. (C) Liver Saa1 mRNA levels. (D-F) ZIP8 KO and littermate controls (fl/fl) were treated with PHZ as described above. n = 6 to 9 mice per group. (D) CBCs were performed on day 4 and day 7 on heparinized blood from ZIP8 KO and fl/fl mice. (E) Day 7 liver Saa1 , liver Nqo1 , and spleen Nqo1 mRNA levels and (F) spleen and serum nonheme iron. (A,C-F) Graphs depict mean and SD; P values determined by 2-tailed unpaired Student t test. SD, standard deviation; WT, wild-type.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Injection, Western Blot, Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: Loss of ZIP8 has no effect on response to iron deficiency in mice. ZIP8 KO mice and littermate controls (fl/fl) were placed on a 4 ppm Fe diet and CBCs were measured at 6, 8, 12, 16, 20, and 24 weeks on the diet. Tissues and serum were harvested at the end of the treatment. n = 8 to 9 mice per group. (A) CBCs from ZIP8 KO and fl/fl mice. (B) Blood ZPP levels. (C) Spleen, liver, and serum nonheme iron levels from iron-deficient ZIP8 KO mice and fl/fl mice. (D) Liver Hamp1 mRNA. (E) Representative western blot for splenic Fpn, Tfr1, and FtnH protein. (A-D) Graphs depict mean and SD. SD, standard deviation; ZPP, zinc protoporphyrin.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Western Blot, Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: ZIP8 is induced by inflammation in human alveolar epithelial cells. (A) Representative images for IHC for ZIP8 (brown) and SP-C (red; AT2 cells) in normal human lung tissue (n = 2 biological replicates). Images are 10× (left) and 40× (right) original magnification. (B-D) A549 cells (human alveolar epithelial cell line) were serum starved for 24 hours, then treated with 50 ng/mL recombinant human IL-6, IFN-γ, IL-1α, IL-1β, TNFα, or epidermal growth factor for 16 hours. n = 1 to 3 independent experiments. (B) Representative western blot for ZIP8 protein expression. (C) SLC39A8 mRNA levels. (D) ZIP8 siRNA knockdown. (E) 16HBE cells (human bronchiolar epithelial cell line) SLC39A8 mRNA levels after treatment for 16 hours. Graphs depict mean and SD. IFN- γ, interferon gamma; KD, ZIP8 siRNA; NT, nontargeting siRNA; SD, standard deviation.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Recombinant, Western Blot, Expressing, Knockdown, Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: ZIP8 transports iron from the airspace into the lung. (A) Slc39a8 mRNA levels of C57BL/6 mouse lung subpopulation cell types. B CD45+: BAL macrophages. L CD45+: lung macrophages. CD31+: endothelial cells. Epcam+: epithelial cells. n = 3 to 4 mice per group. (B) ZIP8 KO mice and littermate controls (fl/fl) were treated with 5 μg of 58 Fe 2+ via RO injection or OP aspiration and harvested after 4 hours for ICP-MS analysis of 58 Fe levels. n = 3 mice per group. (C-D) 16-week-old ZIP8 KO and fl/fl mouse lung (C) nonheme iron levels and (D) Nqo1 mRNA and MDA levels. n = 4 to 10 mice per group. (A-D) Graphs depict mean and SD; P values were determined by 2-tailed unpaired Student t test. MDA, malondialdehyde; SD, standard deviation.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Injection, Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: ZIP8 does not play a significant role in LPS-induced ALI. 16-week-old ZIP8 KO mice and littermate controls (fl/fl) were treated with 15 mg/kg LPS through OP aspiration to induce ALI and harvested after 3 days. A subset of mice in both groups were left untreated for comparison. n = 3 to 7 mice per group. (A) Representative western blot for ZIP8 in lung lysate. (B) Lung nonheme iron levels. (C) BAL protein levels. (D) BAL and lung tissue MPO activity. (E) Lung mitochondrial SOD. (B-E) Graphs depict mean and SD. SD, standard deviation.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Comparison, Western Blot, Activity Assay, Standard Deviation

Journal: Blood Advances

Article Title: A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection

doi: 10.1182/bloodadvances.2022007867

Figure Lengend Snippet: ZIP8 deletion is protective against K pneumoniae infection. ZIP8 KO mice and littermate controls (fl/fl) were infected with 1500 CFU of K pneumoniae via OP aspiration and weighed daily until harvest at 3 days. (A) Percent weight loss in ZIP8 KO and fl/fl mice. n = 3 to 4 mice per group. (B) CFU counts in lung, liver, spleen, and blood after infection. (C) BAL protein levels. (D) BAL and lung tissue MPO activity. (E) Lung mitochondrial and cytosolic SOD. Graphs depict mean and SD; P values were determined by 2-tailed unpaired Student t test. SD, standard deviation.

Article Snippet: Monoclonal mouse anti-ZIP8 antibody (9D4A9) was generated and purified by Genscript using the antigen protein sequence: MHPSEGPELAFSEDVLSVFGANRSLSAAQLGRLLERLGAASQQGALDLGQLHFNQCLSAEDIFSLHGFSNVTQITSSNFSAICPAILQQLNFHPCEDLRKHNAKPSHHHHHH.

Techniques: Infection, Activity Assay, Standard Deviation