Journal: PLoS ONE

Article Title: Clioquinol Synergistically Augments Rescue by Zinc Supplementation in a Mouse Model of Acrodermatitis Enteropathica

doi: 10.1371/journal.pone.0072543

Figure Lengend Snippet: Mice homozygous for the floxed Zip4 gene and positive for the vil-CreERT2 gene ( Zip4 intest KO ) and littermates homozygous for the floxed Zip4 gene but negative for the vil-CreERT2 gene ( control ) were injected for 3 consecutive days with tamoxifen beginning 2 to 3 weeks after weaning and their body weights were monitored thereafter. These mice were fed normal chow and provided access to deionized water after weaning and then fed chow containing CQ and/or provided water containing excess zinc beginning on the indicated days. ( A ) Mice were fed chow containing CQ ( CQ ) or CQ chow plus water containing excess zinc ( CQ/Zn ) beginning 3 days after initiation of the intestine knockout. Only representative mice are shown for the sake of clarity in the figure. ( B ) On d8 after initiation of the intestine knockout mice were provided with water containing excess zinc. ( C ) On d8 after initiation of the intestine knockout these mice were fed chow containing CQ and provided access to drinking water containing excess zinc. Among these mice 6/8 thrived while ( D ) 2 did not thrive and one appeared to be blind in one eye.

Article Snippet: We further speculate that maternal zinc deficiency during pregnancy may predispose some individuals to SMON since we know that maternal heterozygosity of Zip4 +/− predisposes her heterozygous offspring to optic atrophy in the mouse model . Future dose-response studies in this mouse model system might help to determine if there is an optimal concentration of CQ and zinc supplementation that can rescue all of these Zip4 intest KO mice or if there is a different 8- hydroxyquinoline derivative that might be more effective.

Techniques: Control, Injection, Knock-Out

Journal: PLoS ONE

Article Title: Clioquinol Synergistically Augments Rescue by Zinc Supplementation in a Mouse Model of Acrodermatitis Enteropathica

doi: 10.1371/journal.pone.0072543

Figure Lengend Snippet: Zip4 intest KO and control mice (n = 9 per group), as described in the legend to , were fed normal chow and provided access to deionized water after weaning. On d8 after initiation of the knockout, mice were fed chow containing CQ and provided with water containing zinc (+CQ/Zn) . On d22 the CQ/Zn diet was replaced with normal chow and deionized water (−CQ/Zn) and the mice were monitored thereafter. ( A ) Northern blot analysis of intestinal Zip4 mRNA from individual control mice ( Con ) and Zip4 intest KO mice on d8 , d13 and d20 (4 mice per group; one sample is shown for d8, three are shown for d13 and two are shown for d20). ( B ) Body weights of Zip4 intest KO mice. ( C ) Body weights of control mice.

Article Snippet: We further speculate that maternal zinc deficiency during pregnancy may predispose some individuals to SMON since we know that maternal heterozygosity of Zip4 +/− predisposes her heterozygous offspring to optic atrophy in the mouse model . Future dose-response studies in this mouse model system might help to determine if there is an optimal concentration of CQ and zinc supplementation that can rescue all of these Zip4 intest KO mice or if there is a different 8- hydroxyquinoline derivative that might be more effective.

Techniques: Control, Knock-Out, Northern Blot

Journal: PLoS ONE

Article Title: Clioquinol Synergistically Augments Rescue by Zinc Supplementation in a Mouse Model of Acrodermatitis Enteropathica

doi: 10.1371/journal.pone.0072543

Figure Lengend Snippet: Mice were fed chow containing CQ and provided with water containing zinc (CQ/Zn) on d8 and harvested on d13. The small intestines were fixed, paraffin embedded and sections were processed for IHC using an antibody against Ser 10 phosphorylated-histone H3. The number of positive cells per field of view was counted in multiple fields of view (n = 14) at 400X magnification in several sections from 3 mice and the values shown are the mean ± S.E.M. Statistical significance was determined using the Unpaired T-test (two-tailed). ****indicates P<0.0001. Sections from knockout mice fed CQ/Zn that had no detectable Zip4 mRNA in the intestine were examined. Insert : A representative photomicrograph of the small intestine stained with antibody against phosphorylated-histone H3.

Article Snippet: We further speculate that maternal zinc deficiency during pregnancy may predispose some individuals to SMON since we know that maternal heterozygosity of Zip4 +/− predisposes her heterozygous offspring to optic atrophy in the mouse model . Future dose-response studies in this mouse model system might help to determine if there is an optimal concentration of CQ and zinc supplementation that can rescue all of these Zip4 intest KO mice or if there is a different 8- hydroxyquinoline derivative that might be more effective.

Techniques: Two Tailed Test, Knock-Out, Staining

Journal: PLoS ONE

Article Title: Clioquinol Synergistically Augments Rescue by Zinc Supplementation in a Mouse Model of Acrodermatitis Enteropathica

doi: 10.1371/journal.pone.0072543

Figure Lengend Snippet: The small intestines from Zip4 intest KO mice were harvested on d8 after initiation of the knockout. ( A ) Paraffin sections were processed for IHC using antibodies against antigens specific to the secretory cell lineages ( PC : Paneth cell lysozyme , GC : Goblet cell mucin 2 and EEC : Enteroendocrine cell chromogranin A ). Brown color indicates positive immunostaining and the black arrows demarcate these cell types. ( B ) Left hand panels are lower magnification views of sections shown in ( A ). Panels on the right are paraffin sections stained with hematoxylin and eosin ( top right ), processed for IHC using an antibody against mouse fatty acid binding protein ( Fabp2 ) which is enterocyte-specific ( middle right ) or antibody against mouse Sox9 which is expressed in stem cells and Paneth cells ( lower right ). These results were reproduced in multiple sections from 4 Zip4 intest KO mice (sections from 2 mice are shown), none of which had detectable Zip4 mRNA in the intestine.

Article Snippet: We further speculate that maternal zinc deficiency during pregnancy may predispose some individuals to SMON since we know that maternal heterozygosity of Zip4 +/− predisposes her heterozygous offspring to optic atrophy in the mouse model . Future dose-response studies in this mouse model system might help to determine if there is an optimal concentration of CQ and zinc supplementation that can rescue all of these Zip4 intest KO mice or if there is a different 8- hydroxyquinoline derivative that might be more effective.

Techniques: Knock-Out, Immunostaining, Staining, Binding Assay

Journal: PLoS ONE

Article Title: Clioquinol Synergistically Augments Rescue by Zinc Supplementation in a Mouse Model of Acrodermatitis Enteropathica

doi: 10.1371/journal.pone.0072543

Figure Lengend Snippet: The small intestines from Zip4 intest KO mice were harvested on d13 after initiation of the knockout and starting CQ/Zn on d8. Sections from these mice which did not have detectable Zip4 mRNA were examined. ( A: left hand panels ) Paraffin sections were processed for IHC using antibodies against antigens specific to the secretory cell lineages ( PC : Paneth cell lysozyme; GC : Goblet cell mucin 2; and EEC : Enteroendocrine cell chromogranin A ). Brown color indicates positive immunostaining. ( A: Right hand panels ) P araffin sections were stained with hematoxylin and eosin ( top right ), processed for IHC using an antibody against mouse fatty acid binding protein ( Fabp2 ) which is enterocyte specific ( middle right ) or antibody against mouse Sox9 which is expressed in stem cells and Paneth cells ( lower right ). These results were reproduced in multiple sections from 4 Zip4 intest KO mice and the results were indistinguishable from those obtained using sections from control mice treated similarly. ( B ) Northern blot detection of Zip5 mRNA in the intestine from Zip4 intest KO mice on d8 and or d13 fed CQ/Zn. ZIP5 is a basolateral zinc transporter that is most abundant in the crypts but is also detectable on villus enterocytes .

Article Snippet: We further speculate that maternal zinc deficiency during pregnancy may predispose some individuals to SMON since we know that maternal heterozygosity of Zip4 +/− predisposes her heterozygous offspring to optic atrophy in the mouse model . Future dose-response studies in this mouse model system might help to determine if there is an optimal concentration of CQ and zinc supplementation that can rescue all of these Zip4 intest KO mice or if there is a different 8- hydroxyquinoline derivative that might be more effective.

Techniques: Knock-Out, Immunostaining, Staining, Binding Assay, Control, Northern Blot

Journal: PLoS ONE

Article Title: Clioquinol Synergistically Augments Rescue by Zinc Supplementation in a Mouse Model of Acrodermatitis Enteropathica

doi: 10.1371/journal.pone.0072543

Figure Lengend Snippet: The small intestine, liver and pancreas from Zip4 intest KO mice were harvested on d8 after initiation of the knockout and on d13 and d20 after starting CQ/Zn on d8 (3 to 5 mice per group). Only data that showed significant changes are presented. Concentrations (ppm) for liver zinc ( A ), iron ( B ) , copper ( C ), manganese ( D ) , and magnesium ( E ) , and intestine iron ( F ) , were normalize to sulfur concentrations (ppm). Individual tissue samples were analyzed by ICP-MS for multiple elements including these essential metals. Data are expressed as metal/sulfur ratio ± S.E.M. Statistical significance was determined using the Unpaired T-test (two-tailed). Values were considered different if P<0.05. *indicates P<0.05; **indicates P<0.01; ***indicates P<0.001. Our previous studies showed that iron, copper and manganese accumulate in the liver when Zip4 is knocked out in the intestine whereas iron is reduced in the intestine . The symbol (>) on the Y-axes indicates approximate metal/sulfur ratios found in control mice.

Article Snippet: We further speculate that maternal zinc deficiency during pregnancy may predispose some individuals to SMON since we know that maternal heterozygosity of Zip4 +/− predisposes her heterozygous offspring to optic atrophy in the mouse model . Future dose-response studies in this mouse model system might help to determine if there is an optimal concentration of CQ and zinc supplementation that can rescue all of these Zip4 intest KO mice or if there is a different 8- hydroxyquinoline derivative that might be more effective.

Techniques: Knock-Out, Two Tailed Test, IF-P, Control

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: Genes with altered expression in the more aggressive ID8-P1 vs. less aggressive ID8-P0 cells detected by RNAseq

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A) ZIP4 expression in ID8-P0 and ID8-P1 cells from ascites (AS-P1) and peritoneal wall (PW-P1). The relative expression levels were quantified using the ratios of ZIP4/β-actin (detected simultaneously in same blots using two colored fluorescence detection by the Li-Cor system as detailed in Supplementary materials) as shown in the figure. Five different ID8-P1 cell types (from different organs) were tested and all of them had elevated ZIP4. Only representative data are shown. (B-D) PE01, OVCAR3 and T29 cells were starved from serum for 16 hr prior to LPA (10 μM) treatment at different times as indicated. (E-F) PE01 and OVCAR3 were starved from serum for 16 hr prior to LPA treatment for different concentrations as indicated for 6 hr for PE01 and 16 hr for OVCAR3. The optimal LPA concentration and times to induce ZIP4 expression were 5-10 μM LPA and 2-6 hr for PE01 cells and 10 μM LPA and 16-24 hr for OVCAR3 cells. Representative results from ≥ three repetitive experiments are shown.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Fluorescence, Concentration Assay

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A-C) ID8, OVCAR3 and PE01 cells were starved from serum for 16 hr when the confluence was at ~80%. The cells were pre-treated with Ki16425 (10 μM), GW9662 (0.1 to 1 μM), or BrP-LPA (10 μM prior to stimulation with LPA (10 μM) (D) Summery of three independent experiments conducted in PE01 cells. (E-F) Human PE01 cell expressed lower level of PPARγ, which was up-regulated by LPA (10 μM). In PE04 cells, control-vector-transfected and CRISPR PPARγ-KO single clones were seeded into 24-well plate (2×10 5 cells/ well) and cultured for 24 hr. Western-blot analyses were performed to examine the expression levels of PPARγ and ZIP4 in the presence or absence of LPA (10 μM) in PE01 and PE04 cells. Representative results from ≥ three repetitive experiments are shown.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Control, Plasmid Preparation, Transfection, CRISPR, Clone Assay, Cell Culture, Western Blot, Expressing

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A) LPA induced ZIP4 in ID8-P0 and ID8–P1 cells; ID8-P1- Zip4 -KD clone was established using shRNA against Zip4 . (B) ID8-P0 ZIP4 over-expression (OE) cells. (C) PE01-ZIP4-OE cells. (D) ZIP4 -KO clones (K36 and K37) in PE04 cells. (E) ZIP4-OE in either ID8 or PE01 cells increased cell proliferation (48 hr to 72 hr) analyses using the MTT assays; conversely. ZIP4-KO in PE04 cells reduced cell proliferation. The proliferation was presented as fold changes normalized to vector-transfected PE01 cells (as one fold). (F) GW9662 dose-dependently inhibited cell proliferation in ID8-P1 and PE04 cells. (G) PE04-PPARγ-KO clones (R2 and R3) reduced cell proliferation. (H) Z IP4 -KD in ID8-P1 cells and –KO in PE04 cells reduced anoikis-resistance. (I) PE04-Z IP4 –KO cellspresented a reduced colony-formation. (J) Extracellular Zn induced cell death in PE04 cells; ZIP4 -KO reduced Zn toxicity in the 1-10 μM range. Representative results from ≥ three repetitive experiments are shown. * P < 0.05; ** P < 0.01; and *** P < 0.001.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: shRNA, Over Expression, Clone Assay, Plasmid Preparation, Transfection

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A-B) Under the conditions that ZIP4 expression was induced in PE01 and OVCAR3 cells, LPA treatment increased SP % ≥ 2 fold. (C) > 99% of PE04 SP cells were CD44 positive; LPA did not alter the percent of CD44 + cells, but increased cell surface expression in SP cells. (D) LPA increased the percentage of CD44+CD117+ cells in OVCAR3. Cells were starved in all LPA related experiments. (E) ZIP4-KO dramatically reduced SP % in PE04 cells. These cells were not starved from FBS when the FACS analyses were done. Representative results from ≥ three repetitive experiments are shown.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A) PE01-ZIP4-OE increased resistance to CDDP-induced cell death. (B-C) PE04-ZIP4-KO increased cell sensitivity to CDDP- and Dox-induced cell death.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A-B) PE04 and OVCAR3 cells were cultured in ultra-low-attachment plates; spheroid-formation was cell density dependent. Cells were cultured in stem cell conditions (see Methods for details) for 7 days. (C-D) Under the same conditions (1×10 4 /mL in either 24- or 96-well plates), PE04 formed larger or more complete spheroids than PE01. (E) PE04 cells formed more complete spheroids under LPA treatment. (F) ZIP4-KO cells formed fewer and smaller spheroids when compared to control cells. The scale bars in D to I = 400 nm. Representative results from ≥ three repetitive experiments are shown.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Control

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A) . The two known EOC CSC markers, ALDH1 and OCT4 expressed at significantly higher levels in both OVCAR3 and PE04 SP vs. non-SP cells. ZIP4-KO in PE04 cells completely suppressed the high expression of these two genes in SP cells. (B) Only SP cells formed spheroids. When ZIP4 was KO in PE04 cells, even though SP cells could still be isolated using Hoechst-exclusion FACS, but these cells could not form spheroids. The SP portion and non-SP portion were separated by BD SORP FACSAria (BD Biosciences, San Jose, CA) after Hoechst-33342 Staining. 1×10 4 cells per well into Corning 24 Well Plate with low attachment surface under stem cell culture conditions. After 7 days in culture, the SP portion generated spheroids from the PE04/OVCAR3 primary cells, but the non-SP portion cannot form any spheroid. (C) Similar results in spheroid-formation were observed in OVCAR3 SP and non-SP cells.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Isolation, Staining, Stem Cell Culture, Generated

Journal: Oncotarget

Article Title: The novel ZIP4 regulation and its role in ovarian cancer

doi: 10.18632/oncotarget.21435

Figure Lengend Snippet: (A-B) Summary of tumor and ascites development of the control (PE01 and PE04), PE01-ZIP4-OE, and PE04-ZIP4-KO cells injected into NSG mice (n=4-7 in each group). (C) Representative tumor pictures of tumors on the peritoneal wall and the ovary. (D) Representative immunostaining of ZIP4 and Ki67 (a proliferation marker) in the tumor sections derived from wild type and ZIP4 KO PE04 cells.

Article Snippet: Mouse ZIP4 antibody (AF7315) was from R&D Systems (Minneapolis, MN).

Techniques: Control, Injection, Immunostaining, Marker, Derivative Assay

Journal: Diseases of the colon and rectum

Article Title: Epigenomic Characterization of Locally Advanced Anal Cancer: An RTOG 98-11 Specimen Study

doi: 10.1097/DCR.0000000000000160

Figure Lengend Snippet: Annotation of genes with differentially methylated CpG Loci by diameter of primary tumor (≤ 5 cm vs. >5 cm)

Article Snippet: Not reported as target of methylation cg04486382 0.64 0.84 0.20 6.99E-04 1.54E-03 cg19810715 0.64 0.82 0.18 4.44E-04 3.26E-03 NPTX1 Neuronal pentraxin I Methylated in cancer, including colon 65 and cervical cancer 66 , 67 . cg20853771 0.68 0.79 0.11 2.18E-03 2.43E-04 OR2C3 Olfactory receptor, family 2, subfamily C, 3 Not reported as target of methylation or involved in cancer cg20320823 0.56 0.69 0.12 6.99E-04 4.33E-04 PAK1 p21 protein-activated kinase 1 Upregulated in several cancers 68 , 69 ; involved in WNT/β-catenin and RAS pathways; Increased cell motility; interaction with HPV 70 cg24536703 0.62 0.72 0.11 3.98E-04 6.91E-05 PDE3B d Phosphodiesterase 3B, Pro-apoptotic in CML 71 ; associated with cisplatin resistance in head and neck SCC 72 cg21901307 0.24 0.35 0.11 2.72E-04 1.36E-03 PLA2G2A Phospholipase A2, group IIA Regulated by WNT/β-catenin pathway; expression associated with improved survival in gastric 73 and esophageal 74 cancers cg13211559 0.72 0.83 0.12 3.98E-04 1.88E-03 PLEKHG1 Pleckstrin homology domain containing, family G member 1 Oncogene; activation leads to aberrant Rho GTPase targets; Not reported as a target for methylation. cg26852242 0.66 0.80 0.14 8.27E-04 4.47E-04 PLEKHG3 Pleckstrin homology domain containing, family G member 3 Oncogene; activation leads to aberrant Rho GTPase targets; Not reported as a target for methylation. cg11802553 0.66 0.77 0.11 2.43E-04 3.02E-04 PON3 Paraoxonase 3 Imprinted in mouse genome; Over-expressed in several cancers 75 ; anti-apoptotic cg04080282 0.48 0.37 −0.10 4.39E-03 7.39E-04 cg08898155 0.41 0.24 −0.16 3.20E-03 5.54E-04 cg11435506 0.33 0.16 −0.17 2.91E-03 2.10E-04 PPP1R9A Protein phosphatase 1, regulatory subunit 9 Imprinted gene; Not reported to be involved in cancer. cg09724492 0.38 0.52 0.14 4.47E-03 5.00E-04 RERE Arginine-glutamic acid dipeptide (RE) repeats Expression reduced in neuroblastoma and leukemia 76 .

Techniques: Methylation, Infection, Derivative Assay, Expressing, Activity Assay, Sequencing, Ubiquitin Proteomics, Histone Deacetylase Assay, DNA Methylation Assay, Amplification, Activation Assay, Binding Assay, Marker, Membrane, Zinc-Fingers

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Effects of the LQL mutations on human ZIP4 endocytosis in HEK293T cells. ( A ) Topology of hZIP4-HA in the plasma membrane. The LQL segment is located in the IL2 loop. The ECD is shown as a green oval. The HA tag (orange rectangle) is exposed to the extracellular space. ( B ) Effects of substitutions of the residues in the LQL segment on human ZIP4-HA endocytosis. E458 is a generally conserved residue in ZIP4 and thus was tested along with the LQL sequence. The internalized mouse anti-HA antibodies were detected by Western blot using an HRP-conjugated anti-mouse antibody. The total expression levels of ZIP4 variants were detected by an anti-HA antibody. Western blot of anti-β actin was used as loading control. ( C ) Comparison of anti-HA antibody uptake between wild-type ZIP4-HA and its variants using a flow cytometry-based assay. ΔMFI was calculated by subtracting the MFI of the cells incubated with the antibody at 4°C from the MFI of the cells incubated with the antibody at 37°C. The values of ΔMFI were then normalized by setting the value for wild-type ZIP4-HA as 100%. Each column chart shows the data in one of three independent experiments with three biological replicates tested for each condition. The representative histograms on the right show the profiles of 4°C (shaded) and 37°C (open). Statistical analyses were performed using two-tailed Student’s t -test. The P values are 0.0011 (L452A) 0.00099 (L452N), 0.015 (L454I), 0.0012 (L454A), and 0.00033 (L454N), respectively. *: P <0.05; **: P <0.01; ***: P <0.001.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Clinical Proteomics, Membrane, Residue, Sequencing, Western Blot, Expressing, Control, Comparison, Flow Cytometry, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Endocytosis of endogenously expressed ZIP4 in HepG2 cells. ( A ) Internalization of the 1G3B2 anti-ZIP4 mAb by HepG2 cells. Live HepG2 cells were incubated with 1G3B2 mAb at 4°C and 37°C, respectively. After wash, fixation and permeabilization, cells were stained with an Alexa Fluor-568 conjugated anti-mouse antibody. Immunofluorescence images were taken using a confocal microscopy. Representative cells from multiple images were shown. Scale bars indicate 10 μm. ( B ) Analysis of 1G3B2 mAb uptake by flow cytometry. The target proteins were knocked down by siRNA. ΔMFI was calculated by subtracting the MFI of the cells incubated with the antibody at 4°C from the MFI of the cells incubated with the antibody at 37°C. The values of ΔMFI were then normalized by setting the value of the control group as 100%. The column chart shows the data in one of two independent experiments with three biological replicates tested for each condition. The representative results are shown in histogram chart on the right. Statistical analyses were conducted using two-tailed Student’s t -test. The P values are 0.0068 (ZIP4), 0.0013 (CHC), 0.0047 (AP2-α), and 0.0017 (AP2-σ2), respectively. **: P <0.01. ( C ) Knockdown of the target proteins by control siRNA or specific siRNA. The expression levels were analyzed in Western blot using the corresponding antibodies.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Incubation, Staining, Immunofluorescence, Confocal Microscopy, Flow Cytometry, Control, Two Tailed Test, Knockdown, Expressing, Western Blot

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Structural model of the AP2-IL2 complex generated by AlphaFold 3. ( A ) Overall structure ( left ) and zoomed-in view ( right ). AP2 is shown in surface mode and the ZIP4-IL2 peptide (438-494, pink) is in cartoon mode ( left ). 14 of 35 predicted models showed a consistent binding of the LxL motif to a hydrophobic pocket in the σ2 subunit ( right ). L452 and L454 are depicted in stick mode. ( B ) Comparison of the binding of the LxL motif ( left ) and the canonical dileucine motif in CD4 ( middle , PDB: 2JKR). The superimposed structures are shown in the right panel. The residues mutated in this work (V88, L103, N9, R15, and E100 in the σ2 subunit) are underlined.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Identification of the key residues in the AP2-IL2 interaction. ( A ) The importance of the hydrophobic pocket in the σ2 subunit. The σ2 subunit in HepG2 cells was knocked down by siRNA and rescued by overexpressing an siRNA-resistant σ2 subunit or its variants with hydrophobic residues (V88 and L103) substituted with polar/charged amino acids. ΔMFI was calculated by subtracting the MFI of the cells incubated with the antibody at 4°C from the MFI of the cells incubated with the antibody at 37°C. The values of ΔMFI were then normalized by setting the value of the control group as 100%. The representative results are shown in histogram chart ( middle ). Statistical analyses were conducted using two-tailed Student’s t -test. The P values from left to right are 0.00020, 0.00056, 0.0020, 0.031, and 0.0011, respectively. *: P <0.05; **: P <0.01; ***: P <0.001. The expression levels of the wild-type σ2 subunit or its variants were examined by Western blot, and β-actin was used as loading control. ( B ) Examination of the involvement of the electrostatic patch (composed of N9, R15, and E100) in the σ2 subunit. The knockdown and rescue experiments and Western blot were conducted in the same as in (A). The P values from left to right are 0.00043, 0.00027, and 0.0035, respectively. ( C ) Endocytosis of wild-type ZIP4-HA and its variants with changed distance between the two leucine residues in the LxL motif. The constructs were transiently expressed in HEK293T cells and the anti-HA antibody uptake assay was conducted and detected as described in . For A, B , and C , the column chart ( left ) shows the data in one of two independent experiments with three biological replicates tested for each condition.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Incubation, Control, Two Tailed Test, Expressing, Western Blot, Knockdown, Construct

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Cysteine accessibility assay of mouse ZIP4 expressed in HEK293T cells. ( A ) Topology of the cysteine-free TMD of mouse ZIP4 (CysFree-TMD). The amino acid sequence of the IL2 loop is bolded and the residues substituted with cysteines are highlighted in red. The LxL motif is shown in green. Note that a cysteine residue is present at the position of 454 in wild-type ZIP4. ( B ) Cartoon illustration of the experimental procedure. ( C ) Cysteine accessibility assays of eight single cysteine variants. For each variant, an NEM titration was conducted and the red dashed line indicates the concentration of NEM above which the cysteine of interest is 100% labelled by NEM. Cells in control group (Con) were not treated with NEM or mPEG5k. An anti-FLAG antibody was used in Western blot experiments. The shown data are the results of one of two independent experiments with similar results.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Sequencing, Residue, Variant Assay, Titration, Concentration Assay, Control, Western Blot

Journal: bioRxiv

Article Title: Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

doi: 10.1101/2025.05.03.652025

Figure Lengend Snippet: Proposed mechanism of zinc-dependent ZIP4 endocytosis. Cellular zinc availability regulates the conformational states of the IL2 loop, where the LxL motif is located. Under zinc depletion conditions, the LxL motif is less accessible (right); with increased zinc levels, the LxL motif becomes more exposed (left), allowing it to be recruited by AP2 (PDB: 2XA7, orientated in the membrane-bound state) through a direct binding to the hydrophobic pocket in the σ2 subunit (indicated by arrow and zoomed in the inset), where canonical dileucine motifs of cargo proteins bind. For clarity, the ECD of ZIP4 is trimmed. The transport domain of ZIP4 is depicted as a two-domain protein with the scaffold and transport domains shown in green and blue, respectively. Alternating access is achieved by vertical sliding of the transport domain against the static scaffold domain, which is proposed to trigger conformational changes of the IL2 loop. Zinc ions are depicted as grey spheres.

Article Snippet: The pCMV3-based vectors for the expression of N-terminal FLAG-tagged mouse ZIP4 (GenBank ID: 72027, Cat# MG53693 - NF) and the untagged human AP2S1 (σ2 subunit of AP2, GenBank ID: 1175, Cat# HG12478 -UT) in mammalian cells were purchased from Sino Biological.

Techniques: Membrane, Binding Assay