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: 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: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: Single-cell RNA sequencing analysis of perfused lung tissue from naïve and infected WT and Zip8 KO mice ( n = 1 per group). a Unbiased cluster analysis revealed 24 clusters across the four treatment groups. b Analysis of CD86 and CD163 expression revealed that clusters 1, 3, 4, 5, 6, 10, 12, and 17 are populated primarily by macrophages of an M1 phenotype. c Cluster 10 is observed primarily only in the Zip8 KO-infected lung. d Violin Plot of Macrophage markers Adgre1 (F480), Itgam (Cd11b), Itgax (Cd11c), CD68, Fcgr1 (CD64), and SiglecF in Cluster 10 of the infected Zip8 KO mouse lung demonstrates a mixed population of resident and recruited cells. e , f IPA analysis of clusters 1, 3, 4, 5, and 10 reveals that all clusters are defective in phagosome formation and that many pathways associated with M1 characteristics and cell death are prevalent in and unique to cluster 10. g – l FACS analysis of lung tissue identifying total leukocyte, neutrophils, and other myeloid-specific cell subtypes. Also see Supplementary Data , Fig. , and Table .

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: Single Cell, RNA Sequencing, Infection, Expressing

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a Immunostaining of F480 (green), nuclear staining with DAPI (blue), TUNEL staining (red), and GFP-tagged bacteria (white) in naïve and infected (48 h post-infection) WT and Zip8 KO mice lung sections at 63X ( n = 4). Scale bars represent 100 µm. White arrows highlight TUNEL+ macrophages. b Mean fluorescence intensity of GFP-tagged D39 S. pneumoniae is significantly increased in Zip8 KO mice ( n = 4) compared to WT infected mice. c Zip8 KO lungs have higher intracellular bacterial burden 18 h after infection as measured by CFU counts. d Mean fluorescence intensity of TUNEL+ macrophages is significantly increased in infected Zip8 KO mice compared to infected WT mice ( n = 4). e Serum SP-D levels are higher in infected Zip8 KO mice compared to infected WT mice ( n = 7–12). f Comparison of modified mouse clinical assessment scores at baseline, 24, and 48 h after infection. g Comparison of lung tissue Zn levels before and 48 h after infection. Data are represented as mean ± SEM for ( b – d ). Statistical analysis was performed using an unpaired t-test. ( * p < 0.02; ** p < 0.006; **** p < 0.0001). See Supplementary Data and Table .

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: Immunostaining, Staining, TUNEL Assay, Bacteria, Infection, Fluorescence, Comparison, Modification

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a Immunofluorescence imaging of LAMP-1 (red) at 15, 30, and 60 min post-infection. The nucleus is stained with DAPI. Scale bars represent 100 µm. b Quantification of LAMP-1 mean fluorescence intensity. c Zip8 KO BMDMs have higher intracellular bacterial burden 18 h after infection as measured by CFU counts. d DQ-BSA (red) stained BMDMs, non-infected and 18 h post-infection. The nucleus is stained with DAPI. Scale bars represent 50 µm. e Quantification of lysosomal enzymatic activity in Zip8 KO BMDMs is decreased compared to WT 18 h post-infection. f Immunoblot of mature cathepsin B and L, and β-actin proteins from non-infected and 18 h post-infection. g Densitometric analysis of mature cathepsin B and L standardized to Β-actin from noninfected and 18 h after infection. h , i Quantification of cathepsin B ( h ) and L ( i ) activity. Zip8 KO BMDMs have decreased cathepsin B & L activity at baseline and 18 h post-infection. j Immunofluorescence imaging of lysosomes using lysotracker (red) at 15- and 30-min post-infection of D39. The nucleus is stained with DAPI. Scale bars represent 50 µm. k Mean fluorescence intensity of lysotracker staining is decreased in Zip8 KO BMDMs at 15- and 30-min post-infection; however, at 60 min there is no difference compared to WT at the same time points. Data is representative of 2–3 independent experiments and are represented as mean ± SEM. Statistical analysis was performed using an unpaired t -test for graphs ( b , c , e , g , h ). ( * p < 0.05, ** p < 0.009, **** p < 0.0001).

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: Immunofluorescence, Imaging, Infection, Staining, Fluorescence, Activity Assay, Western Blot

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a , b Immunoblot ( a ) and quantification ( b ) of mTOR shows Zip8 KO BMDMs have increased phosphorylation of mTOR compared to WT BMDMs at 1 h post-infection. c Cytoplasmic and nuclear staining of total TFEB in WT BMDMs from FACs analysis. Images include brightfield, TFEB-red, DAPI-stained nucleus, and a merge of TFEB/DAPI. d Zip8 KO BMDMs have less nuclear total TFEB 1 h post-infection. e , f Western analysis similarly reveals a decrease in nuclear TFEB Zip8 KO BMDMs before and after infection. g Pre-treatment with mTOR inhibitor, 250 nM rapamycin for 24 h, reduced bacterial burden and restored clearance in Zip8 KO BMDMs to a level similar to WT BMDMs. h Intracellular Zn concentration is lower in infected Zip8 KO BMDMs compared to infected WT BMDMs. i Pretreatment of WT BMDMs with the Zn chelator TPA (1 µM) for 30 min increased bacterial burden following infection and also further increased bacterial burden in Zip8 KO BMDMs. Data is representative of a minimum of two independent experiments and are represented as mean ± SEM. Statistical analysis was performed by an unpaired t -test for graphs ( b , d , e – g ). ( * p < 0.05, ** p < 0.007, *** p < 0.0006). Also see Supplementary Data and Fig. 4a, b.

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: Western Blot, Phospho-proteomics, Infection, Staining, Concentration Assay

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a Change in metabolite pool sizes across macrophage clusters in WT and Zip8 KO before infection as predicted by metabolic modeling. Statistical analysis was performed using an unpaired t -test for graphs ( a , d ) ( ** p < 0.05). b Change in flux space of Zip8 KO models after adding butyrate before and after infection. Significant changes in flux differences before and after butyrate addition are denoted as mean flux difference ± standard deviation in ( b , e ). c Average change in metabolite pool sizes of butanoate metabolite intermediates of WT and Zip8 KO macrophages before infection. Statistical significance is based on Fold Change, where ( * represents FC > = 1, ** represents FC > = 3, and *** represents FC > = 5). d Change in metabolite pool sizes across macrophage clusters in WT and Zip8 KO post-infection as predicted by the metabolic models. Statistical analysis was performed using an unpaired t -test for graphs ( a , d ) ( ** p < 0.05). e Change in flux space of Zip8 KO models after adding butyrate in both post-infection conditions. Significant changes in flux differences before and after butyrate addition are denoted as mean flux difference ± standard deviation. f Average change in metabolite pool sizes of butanoate metabolite intermediates of WT and Zip8 KO macrophages after infection. Statistical significance is based on fold change ( * represents FC = 1, ** represents FC > = 3, and *** represents 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: Infection, Standard Deviation

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a PBA-treated and untreated WT and Zip8 KO mice were infected with 4 × 10 8 CFU S. pneumoniae, and the pneumonia clinical assessment score was assessed up to 48 h post-infection. b – f PBA-treated and untreated WT and Zip8 KO mice were infected with 4 × 10 8 CFU S. pneumoniae , and lungs were harvested and examined at 48 h post-infection and at baseline for total BAL cell count ( b ), total macrophage count ( c ), total neutrophil count ( d ), BAL protein ( e ), and CXCL1 levels ( f ). ( n = 10-14 per group). g – l PBA-treated and untreated WT and Zip8 KO mice were infected with 4 × 10 8 CFU S. pneumoniae, and lungs were harvested and examined by FACS at 48 h post-infection and at baseline for total leukocytes, neutrophils, and other myeloid-specific subsets. Data are represented as mean ± SEM. Statistical analysis was performed by an unpaired t -test for graphs ( a – f ). ( * p < 0.05, ** p < 0.004, *** p < 0.008).

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, Cell Characterization

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a Immunostaining of F480 (green), nuclear staining with DAPI (blue), TUNEL (red), and D39 (white) staining in PBS or PBA-treated WT infected mice (upper left and lower left panels) and PBS or PBA-treated infected Zip8 KO mice at 48 h following infection. Lung sections magnified at 63× ( n = 10). Scale bars represent 100 µm. b Quantification of imaging of WT and Zip8 KO mouse lungs at 48 h post-infection revealed a significant decrease in S. pneumoniae (D39) in Zip8KO mouse lungs following PBA treatment compared to vehicle control counterparts . c Similarly, quantification of Colony Forming Unit counts obtained from WT and Zip8KO mouse lungs at 48 h post-infection revealed a significant decrease in S. pneumoniae (D39) in Zip8KO mouse lungs following PBA treatment compared to vehicle control counterparts . d Quantification of apoptotic macrophages following PBA treatment significantly decreased in TUNEL-positive macrophages in infected WT and even more so in infected Zip8 KO mice ( n = 4). e Serum SP-D levels decreased in PBA-treated Zip8 KO post-infection. ( n = 9-12). f PBA had no effect on zinc levels in lung tissue post-infection. ( n = 9-12). Data are represented as mean ± SEM. Statistical analysis was performed by an unpaired t -test. ( * p < 0.05, ** p < 0.006 for graphs ( b – e ).

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: Immunostaining, Staining, TUNEL Assay, Infection, Imaging, Control

Journal: Communications Biology

Article Title: ZIP8 loss impairs macrophage-mediated phagolysosomal removal of bacteria and is overcome by butyrate supplementation

doi: 10.1038/s42003-025-09504-8

Figure Lengend Snippet: a Treatment with PBA post-differentiation does not improve bacterial clearance in Zip8 KO BMDMs. b PBA supplementation during differentiation improves bacterial clearance in Zip8 KO BMDMs. c , d PBA normalizes the phosphorylation of mTORC1 in Zip8 KO BMDMS, similar to that of WT BMDM levels following infection. e PBA treatment increases TFEB nuclear localization in Zip8 KO BMDMs following infection. f Immunofluorescence images of DQ-BSA-stained WT and Zip8 KO BMDMs with and without PBA supplementation before and after infection. g PBA treatment increases lysosome enzyme activity in Zip8 KO BMDMs before and after infection. h PBA treatment increases Cathepsin L activity in Zip8 KO BMDMs following infection. The data represented are three independent experiments, and represented as mean ± SEM. Statistical analysis was performed by an unpaired t -test for graphs ( a – f ). ( * p < 0.05, ** p < 0.009, *** p < 0.0007).

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: Phospho-proteomics, Infection, Immunofluorescence, Staining, Activity Assay