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: 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: European Journal of Medical Research

Article Title: C-type lectin domain family 3 member B (CLEC3B) inhibits triple-negative breast cancer chemoresistance via inducing ferroptosis

doi: 10.1186/s40001-025-02855-2

Figure Lengend Snippet: CLEC3B is downregulated in TNBC tissues and associated with poor prognosis of TNBC patients. A . The expression of CLEC3B mRNA in normal mammary tissues, noTNBC and TNBC. B . Kaplan–Meier analyses of overall survival of CLEC3B in TNBC patients and TNBC patients with chemotherapy. C . qRT-PCR and western blotting assay of the expression of CLEC3B in TNBC patients with or without recurrence after chemotherapy. These experiments were replicated three times. D . qRT-PCR assay of the expression of CLEC3B in TNBC patients with or without recurrence after chemotherapy. These experiments were replicated three times. Data represents the mean ± SD, * p < 0.05, *** p < 0.001

Article Snippet: The following antibodies were used: anti-CLEC3B antibody (cat. 20311–1-AP, Proteintech, Rosemont, IL, USA), anti-SLC39A14 (cat. ab106568, Abcam, Cambridge, UK), anti-SLC39A8 (cat. SAB3500598, Sigma, St. Louis, MO, USA), and anti-GAPDH antibody (cat. ab8245, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: European Journal of Medical Research

Article Title: C-type lectin domain family 3 member B (CLEC3B) inhibits triple-negative breast cancer chemoresistance via inducing ferroptosis

doi: 10.1186/s40001-025-02855-2

Figure Lengend Snippet: CLEC3B promotes CDDP chemosensitivity. A . Western blotting confirming the effect of CLEC3B overexpression and knockdown. B . The effect of CLEC3B on the IC50 of CDDP. C . MTT assay of the effect of CLEC3B on TNBC cell proliferation under CDDP treatment. D . Colony formation assay of the effect of CLEC3B on TNBC cell proliferation under CDDP treatment (upper). TUNEL assay of the effect of CLEC3B on TNBC apoptosis under CDDP treatment (bottom). All experiments were replicated three times. Data represents the mean ± SD, * p < 0.05

Article Snippet: The following antibodies were used: anti-CLEC3B antibody (cat. 20311–1-AP, Proteintech, Rosemont, IL, USA), anti-SLC39A14 (cat. ab106568, Abcam, Cambridge, UK), anti-SLC39A8 (cat. SAB3500598, Sigma, St. Louis, MO, USA), and anti-GAPDH antibody (cat. ab8245, Abcam, Cambridge, UK).

Techniques: Western Blot, Over Expression, Knockdown, MTT Assay, Colony Assay, TUNEL Assay

Journal: European Journal of Medical Research

Article Title: C-type lectin domain family 3 member B (CLEC3B) inhibits triple-negative breast cancer chemoresistance via inducing ferroptosis

doi: 10.1186/s40001-025-02855-2

Figure Lengend Snippet: CLEC3B induces TNBC ferroptosis under CDDP. A . Cytotoxicity assay of vector control and CLEC3B-overexpressing TNBC cells treated or not treated with CDDP in combination with the apoptosis inhibitor Z-VAD-FMK (VAD), the ferroptosis inhibitor ferrostatin-1 (Fer), the pyroptosis inhibitor Ac-DMPD/DMLD-CMK (DMPD/DMLD) the necroptosis inhibitor necrostatin (Nec) or autophagy inhibitor 3-methyladenine (3-me). B and C . The effect of CLEC3B on Fe 2+ current and MDA level in TNBC cells under CDDP treatment. All experiments were replicated three times. Data represents the mean ± SD, * p < 0.05

Article Snippet: The following antibodies were used: anti-CLEC3B antibody (cat. 20311–1-AP, Proteintech, Rosemont, IL, USA), anti-SLC39A14 (cat. ab106568, Abcam, Cambridge, UK), anti-SLC39A8 (cat. SAB3500598, Sigma, St. Louis, MO, USA), and anti-GAPDH antibody (cat. ab8245, Abcam, Cambridge, UK).

Techniques: Cytotoxicity Assay, Plasmid Preparation, Control

Journal: European Journal of Medical Research

Article Title: C-type lectin domain family 3 member B (CLEC3B) inhibits triple-negative breast cancer chemoresistance via inducing ferroptosis

doi: 10.1186/s40001-025-02855-2

Figure Lengend Snippet: CLEC3B promotes ferroptosis via interacting with SLC39A8 and SLC39A14. A . CoIP assay of the interaction of CLEC3B and SLC39A8 or SLC39A14 in HCC1937 cells. B . Western blotting assay confirming the effect of SLC39A8 and SLC39A14 overexpression and knockdown in HCC1937 cells transduced with shRNAs against SLC39A8 and SLC39A10, respectively. C and D . The effect of knockdown of SLC39A8 and SLC39A14 on Fe 2+ current and MDA level in CLEC3B-overexpressing TNBC cells under CDDP treatment. All experiments were replicated three times. Data represents the mean ± SD, * p < 0.05

Article Snippet: The following antibodies were used: anti-CLEC3B antibody (cat. 20311–1-AP, Proteintech, Rosemont, IL, USA), anti-SLC39A14 (cat. ab106568, Abcam, Cambridge, UK), anti-SLC39A8 (cat. SAB3500598, Sigma, St. Louis, MO, USA), and anti-GAPDH antibody (cat. ab8245, Abcam, Cambridge, UK).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Over Expression, Knockdown, Transduction

Journal: European Journal of Medical Research

Article Title: C-type lectin domain family 3 member B (CLEC3B) inhibits triple-negative breast cancer chemoresistance via inducing ferroptosis

doi: 10.1186/s40001-025-02855-2

Figure Lengend Snippet: SLC39A8 and SLC39A14 knockdown reverse the role of CLEC3B overexpression in CDDP chemosensitivity. A . MTT assay of the effect of Fer treatment of knockdown of SLC39A8 and SLC39A14 on CLEC3B overexpression on chemosensitivity. B . Colony formation assay of the effect of Fer treatment or knockdown of SLC39A8 and SLC39A14 on CLEC3B overexpression on chemosensitivity (upper). TUNEL assay of the effect of Fer treatment or knockdown of SLC39A8 and SLC39A14 on CLEC3B overexpression on chemosensitivity (bottom). All experiments were replicated three times. Data represents the mean ± SD, * p < 0.05

Article Snippet: The following antibodies were used: anti-CLEC3B antibody (cat. 20311–1-AP, Proteintech, Rosemont, IL, USA), anti-SLC39A14 (cat. ab106568, Abcam, Cambridge, UK), anti-SLC39A8 (cat. SAB3500598, Sigma, St. Louis, MO, USA), and anti-GAPDH antibody (cat. ab8245, Abcam, Cambridge, UK).

Techniques: Knockdown, Over Expression, MTT Assay, Colony Assay, TUNEL Assay

Journal: Cardiovascular Diabetology

Article Title: Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells

doi: 10.1186/s12933-024-02224-z

Figure Lengend Snippet: Slc39a14 and Slc39a8 are involved in the altered iron metabolism observed in vascular calcification. Venous blood was collected and serum ferrous iron levels were determined using an iron assay kit ( A ). Serum calcium and alkaline phosphatase (ALP) concentrations were measured and normalised to protein levels ( B and C ). Relative RNA levels of Runx2, Slc39a14, Slc39a8, DMT1, and TFRC were analysed by qPCR and normalised; *P < 0.05, **P < 0.01 ( D ). Total aortic proteins were used to determine the expression of Slc39a14, Slc39a8, and TFRC by western blotting (*P < 0.05, **P < 0.01, E and F ). The expression of Slc39a14, Slc39a8, and TFRC in aortic sections was examined by immunohistochemical (IHC) staining. Black arrow head indicates the medial layer of artery. Scale bar: 50 µm ( G ). The expression levels of Slc39a14, Slc39a8, and TFRC were determined through immunofluorescence staining. White arrow head indicates the medial layer of artery. Scale bar: 20 µm ( H )

Article Snippet: Western blotting analyses were performed using specific antibodies against β-actin (Affinity, AF7018), BMP2(Abcam, ab284387), RUNX2 (CST, D1L7F), FTH1 (Abclonal, A19544), FTL (Abclonal, A18051), α-SMA (Proteintech, 67735-1-lg), Slc39a8 (Proteintech, 20459-1-AP), Slc39a14 (Affinity,DF14224) and TFRC (Proteintech, 66180-1-Ig).

Techniques: Iron Assay, Expressing, Western Blot, Immunohistochemical staining, Immunohistochemistry, Immunofluorescence, Staining