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: Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

doi: 10.1101/2025.10.23.682519

Figure Lengend Snippet: Xenopus oocytes injected either with ( A, B ) human ZIP14 or ( E, F ) human ZIP8 cRNA were incubated with either ( A, E ) ⁶⁵Zn or ( B, F ) ⁵⁵Fe in the presence of ( A, E ) 50 µM or ( B, F ) 10 µM PPTD for 30 min at 22 °C. Radioactivity was measured as described under Methods . Data are normalized against DMSO-treated transporter-expressing controls, and presented as mean ± standard deviation (S.D.) (n=3 independent experiments). Similarly, (C, D) TREx-hZIP14 or (G, H) TREx-hZIP8 cells were treated with Tet (1 μg/mL) for 24 h, followed by incubation either with ( C, G ) ⁵⁴MnCl₂ or (D, H) ¹⁰⁹CdCl₂ in the presence of PPTD at the indicated concentrations for 1 h. Radioactivity was measured as described under Methods . The data (mean + SD) represent results from three independent experiments. Statistical significance was determined using Student’s t -test (A, B, E, F) or one-way ANOVA followed by Dunnett’s post hoc test (C, D, G, H). (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ***** p < 0.0005).

Article Snippet: The following primary antibodies were used: anti-human ZIP14 (generated previously ), anti-human ZIP8 (Alomone Labs, #AZT-008), and anti-β-actin (Cell Signaling Technology, #3700S).

Techniques: Injection, Incubation, Radioactivity, Expressing, Standard Deviation

Journal: Frontiers in Physiology

Article Title: The Allelic Variant A391T of Metal Ion Transporter ZIP8 (SLC39A8) Leads to Hypotension and Enhanced Insulin Resistance

doi: 10.3389/fphys.2022.912277

Figure Lengend Snippet: Murine ZIP8 homology model, metal ion transport and plasma membrane expression of human ZIP8 WT and A391T mutant in transiently transfected HEK293T cells. (A) Left panel: 3D structure of mouse ZIP8 based on the bbZIP X-ray structure . Right panel: Diagram indicating the disposition of C and N-terms and the colouring and numbering of the 8 Transmembrane Helix (TMH). (B) Left panel: Representative fluorescence microscopy images of intracellular Zn 2+ (10 μM) accumulation. Right panel: Normalized values from six independent experiments ( n = 22–51) are represented individually. (C) Left panel: Representative experiment showing the change on fluorescence intensity as result of intracellular Cd 2+ (10 μM) accumulation. Right panel: Normalized values from four independent experiments ( n = 2–30) are represented individually. (D) Left panel: Representative experiment showing 55 Fe 2+ transport kinetics [0.1–10 μM]. Middle panel: Normalized values of iron transport (10 μM) obtained from five independent experiments ( n = 27–40) are represented individually. Right panel: Iron transport (1 μM) in the presence of an excess of different divalent metals (Zn 2+ , Cd 2+ , Co 2+ , Cu 2+ , Mn 2+ and Ba 2+ ) (10 μM). Normalized values from independent experiments ( n = 2) are represented individually. (E) Representative blot showing the plasma membrane surface protein expression determined using an anti-HA monoclonal antibody. Normalized results obtained from three independent experiments, performed in duplicate. (F) Left panel: Biotin content of each sample as loading control. Right panel: Plasma membranes expression of Na + /H + exchanger (NHE-1) and lack of β-actin expression are shown as control of membrane surface samples purity. All the data are mean ± SEM of the indicated number of biological replicates. Statistical differences between groups were assessed using either t -test or Mann-Whitney U according to the sample distribution. Significance was set at p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. NT, non-transfected; EV, empty vector.

Article Snippet: The human SLC39A8 (ZIP8) ORF (NCBI Reference Sequence: NM_022154.5) containing a C-terminal TurboGFP tag, was cloned into the pCMV6-AC-GFP vector (OriGene #RG204200).

Techniques: Expressing, Mutagenesis, Transfection, Fluorescence, Microscopy, MANN-WHITNEY, Plasmid Preparation

Journal: Frontiers in Physiology

Article Title: The Allelic Variant A391T of Metal Ion Transporter ZIP8 (SLC39A8) Leads to Hypotension and Enhanced Insulin Resistance

doi: 10.3389/fphys.2022.912277

Figure Lengend Snippet: ZIP8KI mice express less ZIP8 protein. (A) Sequencing analysis for A393T mutation in the Slc38A9 mice. The 1177G>A mutation in exon 8 results in an exchange of alanine (Ala) to threonine (Thr, blue). For detection of the mutated allele in the genome the SspI restriction site is deleted by a silent exchange of 1185T>C. (B) Genotyping for the detection of the A393T mutation in the Slc39A8 locus. DNA extracted from ear biopsies of mice. The expected PCR signal of 503 bp is cut into two fragments of 371 and 132 bp in the wild type, and the mutant allele remain uncut fragment of 503 bp. The homozygote mouse shows the expected cleavage products, demonstrating introduction of the mutation into the genome. (C) Relative mRNA expression of slc39a8 in lung and (D) kidney of WT and ZIP8KI male mice normalized to actin under StD diet ( n = 8). (E) Western blot analysis and quantification of ZIP8 protein expression in lung and (F) kidney membrane extracts in WT and ZIP8KI ( n = 5–15) Means ± SEM of animals in each experimental condition. ∗ p < 0.05 comparisons made between WT and ZIP8KI mice.

Article Snippet: The human SLC39A8 (ZIP8) ORF (NCBI Reference Sequence: NM_022154.5) containing a C-terminal TurboGFP tag, was cloned into the pCMV6-AC-GFP vector (OriGene #RG204200).

Techniques: Sequencing, Mutagenesis, Expressing, Western Blot

Journal: Nutrients

Article Title: ZIP8 Is Upregulated in the Testis of Zip14 -/- Mice

doi: 10.3390/nu16213575

Figure Lengend Snippet: Validation of antibodies used in immunoblotting analyses. ( A ) Immunoblotting analysis of ZIP8 expression in lung tissues from wild-type (WT) and tamoxifen-induced Zip8 knockout ( Zip8 -iKO) mice. The anti-ZIP8 primary antibody shows specific detection of ZIP8 protein in WT lungs, with no signal observed in Zip8 -iKO lungs. ( B ) Immunoblotting analysis of ZIP14 expression in liver tissues from WT and Zip14 knockout ( Zip14 -/- ) mice. ( C ) Immunoblotting analysis of ZnT10 expression in liver tissues from WT and Znt10 knockout ( Znt10 -/- ) mice. These results confirm the specificity of the antibodies used for ZIP8, ZIP14, and ZnT10 in immunoblotting.

Article Snippet: To generate inducible Zip8 knockout ( Zip 8-iKO) mice, animals carrying the Zip8 conditional allele ( Zip8 flox/flox ) (purchased from Taconic, Germantown, NY, USA) were crossbred with mice expressing the Cre-ERT2 (tamoxifen-inducible estrogen receptor fusion protein) under the control of the ubiquitin promoter ( Ubc -Cre) (obtained from the Jackson Laboratory, Bar Harbor, ME, USA).

Techniques: Biomarker Discovery, Western Blot, Expressing, Knock-Out

Journal: Nutrients

Article Title: ZIP8 Is Upregulated in the Testis of Zip14 -/- Mice

doi: 10.3390/nu16213575

Figure Lengend Snippet: Expression of manganese transporters in the testis. ( A ) Immunoblotting analysis of ZIP8 expression in testes from WT and Zip8 -iKO mice. A distinct ZIP8 band is observed in WT testis but is absent in Zip8 -iKO testis, confirming the expression of ZIP8 in the testis. ( B ) Immunoblotting analysis of ZIP14 expression in WT and Zip14 -/- testes. No detectable signal for ZIP14 is observed, suggesting minimal or no expression of ZIP14 in the testis. ( C ) Immunoblotting analysis of ZnT10 expression in WT and Znt10 -/- testes. Similar to ZIP14, no detectable ZnT10 signal is observed, indicating that ZnT10 is either not expressed or is below the detection limit in testicular tissue.

Article Snippet: To generate inducible Zip8 knockout ( Zip 8-iKO) mice, animals carrying the Zip8 conditional allele ( Zip8 flox/flox ) (purchased from Taconic, Germantown, NY, USA) were crossbred with mice expressing the Cre-ERT2 (tamoxifen-inducible estrogen receptor fusion protein) under the control of the ubiquitin promoter ( Ubc -Cre) (obtained from the Jackson Laboratory, Bar Harbor, ME, USA).

Techniques: Expressing, Western Blot

Journal: Nutrients

Article Title: ZIP8 Is Upregulated in the Testis of Zip14 -/- Mice

doi: 10.3390/nu16213575

Figure Lengend Snippet: ZIP8 protein expression in the testes of Zip14 -/- mice increases. ( A ) Immunoblotting analysis of ZIP8 protein levels in testes from WT and Zip14 -/- mice. ( B ) Quantification of ZIP8 protein expression, normalized to β-Actin, reveals a significant increase in ZIP8 levels in Zip14 -/- mice. Data are expressed as mean ± SD ( n = 5). * p < 0.05.

Article Snippet: To generate inducible Zip8 knockout ( Zip 8-iKO) mice, animals carrying the Zip8 conditional allele ( Zip8 flox/flox ) (purchased from Taconic, Germantown, NY, USA) were crossbred with mice expressing the Cre-ERT2 (tamoxifen-inducible estrogen receptor fusion protein) under the control of the ubiquitin promoter ( Ubc -Cre) (obtained from the Jackson Laboratory, Bar Harbor, ME, USA).

Techniques: Expressing, Western Blot

Journal: Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS)

Article Title: Imbalance in Zinc Homeostasis Enhances Lung Tissue Loss Following Cigarette Smoke Exposure

doi: 10.1016/j.jtemb.2020.126483

Figure Lengend Snippet: (A) Comparison of average chord length measured across alveolar tissue obtained from Zip8KO mice or corresponding WT littermate controls. Groups of mice were either exposed to control room air or cigarette smoke (CS) for a period of four months. Data are representative of two independent experiments with a minimum of six mice per treatment group. (B) Representative photomicrographs of H&E stained lung sections obtained from each treatment group. Ablation of ZIP8 expression resulted in increased alveolar tissue loss following 4 months of CS exposure over 4 months when compared to WT, CS-exposed mice. (Data shown are a combination from two separate experiments with a minimum of twelve animals per treatment group. Bar graphs depict SD with error bars. Significance values for indicated comparisons by one-way ANOVA and Tukey’s post test; * designates p < 0.05).

Article Snippet: ZIP8 flox/flox mice were crossed to myeloid cell specific LysMcre (The Jackson Laboratory) ( 20 )to generate the conditional ZIP8KO . . Bronchoalveolar Lavage, Leukocyte Enumeration, and Cytokine Analysis Lungs were lavaged three times with 1 ml of 1× Hank’s balanced salt solution.

Techniques: Comparison, Staining, Expressing

Journal: Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS)

Article Title: Imbalance in Zinc Homeostasis Enhances Lung Tissue Loss Following Cigarette Smoke Exposure

doi: 10.1016/j.jtemb.2020.126483

Figure Lengend Snippet: Transgenic ZIP8 overexpressing (Zip8tg) mice were exposed to CS daily for a period of 7 months. A time dependent increase in lung Cd content occurred over 7 months (A). Lung Cd deposition was significantly increased in Zip8tg compared to WT mice at 7 months (B), with no difference in lung Zn between the two groups (C). Alveolar loss at 7 months was increased in Zip8tg mice compared to WT, CS-exposed counterparts (D). (B) Both WT and Zip8tg animals exhibited increased lung ROS content and increased kidney Cd content (F). (Data shown are representative from two separate experiments with a minimum of six animals per treatment group. Bar graphs depict SD with error bars. Significance values for indicated comparisons by one-way ANOVA and Tukey’s post test; * designates p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: ZIP8 flox/flox mice were crossed to myeloid cell specific LysMcre (The Jackson Laboratory) ( 20 )to generate the conditional ZIP8KO . . Bronchoalveolar Lavage, Leukocyte Enumeration, and Cytokine Analysis Lungs were lavaged three times with 1 ml of 1× Hank’s balanced salt solution.

Techniques: Transgenic Assay