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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

Techniques: Recombinant, Western Blot, Expressing, 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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

Techniques: 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: For IHC for ZIP8 and SP-C in human lung tissue, antigen retrieval was performed by boiling in Tris-EDTA (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 9.0) for 10 min. IHC was performed using ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7451) and ImmPRESS-AP Horse Anti-Mouse IgG Polymer Kit, Alkaline Phosphatase (MP-5402) according to the manufacturer’s instructions, with rabbit anti-ZIP8 rabbit antibody (Sigma HPA038833) and mouse anti-SP-C antibody (Santa Cruz, sc-518029) used as the primary antibody at 200 ng/mL.

Techniques: Infection, Activity Assay, Standard Deviation