antibody against zip14 pa5-87880  (Thermo Fisher)

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: A Schematic workflow of the high-throughput screening to identify ZIP14 inhibitors. B Chemical structure of 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD).

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: High Throughput Screening Assay

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: 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: A Structural predictions of ZIP14-PPTD complexes using both monomeric (left) and dimeric (right) forms of human ZIP14 by AlphaFold3. Magenta circles indicate the predicted PPTD binding sites. Protein models are colored by structure prediction confidence estimated by predicted Local Distance Difference Test (pLDDT) (dark blue, pLDDT >90, light blue, pLDDT of 90-70, yellow, pLDDT of 70-50, orange, pLDDT <50). B Structural overlay of the ZIP14-PPTD and ZIP14-zinc complexes, predicted using AlphaFold3. PPTD and zinc bind in close proximity within the dimerized ZIP14 protein. magenta: PPTD, purple: zinc, blue: His347 and His380 residues. C Prediction of human ZIP14 amino acid residues interacting with PPTD by Protein–Ligand Interaction Profiler and AlphaFold3 (Supplementary Table 5). magenta: PPTD, green dot line: pi-stacking, yellow dot line: salt bridges, gray dot line: hydrophobic interactions, blue line: hydrogen bond. D Asp348 is essential for ZIP14-mediated zinc transport. Xenopus oocytes were injected with human ZIP14 (WT), ZIP14 (D348L), ZIP14 (D348N) cRNA, or water (wi, control). The uptake assay was performed with 10 μM ZnSO 4 including a trace amount of radioactive tracer in the presence of 50 μM PPTD for 30 min at 22 °C. Radioactivity was measured as described under Methods . Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. (*** p < 0.001). All data are shown as mean ± S.E.M. (n= 6 to 9 oocytes).

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: Binding Assay, Injection, Control, Radioactivity, Comparison

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: A Comparison of structural simulations of the human ZIP14 (WT)-PPTD complex (upper) and the ZIP14 (S343Y)-PPTD complex (lower). Substitution of Serine 343 with Tyrosine (S343Y) reduces the size of the PPTD-binding pocket within ZIP14, as indicated by the dotted red circle in the mutant structure. magenta: PPTD. B Serine 343 is essential for ZIP14-mediated zinc transport. Xenopus oocytes were injected either with human ZIP14 (WT), ZIP14 (S343Y) cRNA, or water (wi, control). The uptake assay was performed with 10 μM ZnSO 4 including a trace amount of radioactive tracer in the presence of 50 μM PPTD. Radioactivity was measured as described under Methods . All data are shown as mean ± S.E.M. (n= 6 to 9 oocytes). C Structural overlay of five modeled complexes of PPTD bound to human ZIP14 with aspartic acid to leucine substitution at position 443 (D443L). In the four models, PPTD consistently occupies an internal binding region of ZIP14 (indicated by the blue arrow), while one model shows PPTD associating with the protein surface (the red arrow). This variation suggests that the D443L mutation may disrupt the stable binding of PPTD to ZIP14. D Aspartic acid at position 443 contributes to the sensitivity of ZIP14 to PPTD. After preincubation with indicated concentrations of PPTD, Xenopus oocytes injected with cRNA encoding wild-type human ZIP14 (WT) or the D443L mutant were incubated with 10 μM ZnSO 4 including a trace amount of radioactive tracer in the presence of the same concentrations of PPTD for 30 min at 22 °C. The IC₅₀ values for ZIP14 WT and D443L were 8.9 μM and 19.0 μM, respectively. Radioactivity and IC₅₀ values were determined as described under Methods . E The ZIP14 D443L mutant retains transport activity but loses sensitivity to PPTD. Xenopus oocytes were injected with cRNA encoding either wild-type human ZIP14 (WT) or the D443L mutant. The uptake assay was performed with 10 μM ZnSO 4 including a trace amount of radioactive tracer in the presence of 20 μM PPTD. Radioactivity was measured as described under Methods . Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (*** p < 0.001). All data are shown as mean ± S.E.M. (n= 6 to 9 oocytes).

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: Comparison, Binding Assay, Mutagenesis, Injection, Control, Radioactivity, Incubation, Activity Assay

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: A Schematic overview of the experimental workflow for PPTD administration to the mouse model of cancer cachexia. B Effects of PPTD administration on body weight loss in the cancer cachexia model. Mice were administered PPTD via drinking water at either 0.1 g/L (low-dose) or 1.0 g/L (high-dose). C PPTD extends survival in the cancer cachexia model. Mice were administered 0.1 g/L (low dose, blue line) or 1.0 g/L (high dose, red line) PPTD through drinking water. Survival was analyzed using the Kaplan-Meier method. Black line: water-treated control group. Survival curves were compared using the Kaplan–Meier method and log-rank test ( p < 0.05 is considered significant). D PPTD delays the onset of cachexia symptoms. Mice were administered 0.1 g/L (low-dose) or 1.0 g/L (high-dose) PPTD via drinking water. The day on which each group first exhibited ≥10% body weight loss or death is shown. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test (* p < 0.05). E PPTD improves locomotor activity in a cancer cachexia model. The locomotor of individual mice (n = 3 per group) was recorded on the 16th day of PPTD administration and analyzed using ImageJ (Supplementary Movie 7), as described under Methods . The results are presented as box plots with gray and blue boxes representing the H₂O control group and the PPTD-treated group (0.1 g/L), respectively. Each point represents an individual mouse. Statistical significance was assessed using Welch’s t-test to compare total distances between groups (* p < 0.05). F Working model: Inflammatory stimuli induce ZIP14 expression, promoting metal-induced cytotoxicity that contributes to cancer cachexia (left). The ZIP14 inhibitor PPTD alleviates key features of cancer cachexia (right) and may contribute to improving patients’ quality of life (QOL) (bottom).

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: Control, Activity Assay, Expressing

Journal: Cancer Medicine

Article Title: Dihydroartemisinin Sensitizes Lung Cancer Cells to Cisplatin Treatment by Upregulating ZIP14 Expression and Inducing Ferroptosis

doi: 10.1002/cam4.70271

Figure Lengend Snippet: Decreased expression of ZIP14 contributed to the tolerance of DHA&DDP‐induced ferroptosis. (A) The qPCR analysis of ZIP14, ZIP8, and DMT1 levels in A549 cells treated with DHA (20 μM) and DDP (10 μM) alone or in combination for 48 h, mean ± SD, n = 3. (B) Immunofluorescence imaging of ZIP14 (red) and DAPI (blue) under mono or combined treatment of DHA (20 μΜ) and DDP (10 μΜ) in A549 cells for 48 h. Scale bars: 100 μm. (C) Western blot was used to detect the expression of ZIP14 proteins in A549 and H1975 cells treated with DHA (20 μΜ for A549 or 40 μΜ for H1975) and DDP (10 μΜ) alone or in combination for 48 h. (D) The level of ZIP14 mRNA was measured by qPCR after RNA interference using shRNA in A549 cells, mean ± SD, n = 3. (E) Western blot was used to detect the expression ZIP14 proteins in A549 cells treated with shZIP14. (F) Membrane‐associated ZIP14 protein levels in shNC or shZIP14 A549 cells were measured using flow cytometry. (G) The viability of shNC or shZIP14 A549 cells treated with DHA (20 μM) and DDP (10 μM) for 48 h by CCK‐8 assay, mean ± SD, n = 3. (H) Western blot was used to detect the expression of the ferroptosis‐associated proteins in shNC or shZIP14 A549 cells treated with DHA (20 μM) and DDP (10 μM) for 48 h. (I) Flow cytometry was used to evaluate changes in ROS (left) and ferrous iron (right) levels in shNC or shZIP14 A549 cells treated with DHA (20 μM) and DDP (10 μM) for 48 h. *** p < 0.001.

Article Snippet: Antibody against ZIP14 (Affinity, DF14224) was obtained from Affinity (Affinity Biosciences, Jiangsu, China) for flow cytometry and immunofluorescence, while the antibody against ZIP14 (Abclonal, A10413) was obtained from Abclonal (Wuhan, China) for western blot.

Techniques: Expressing, Immunofluorescence, Imaging, Western Blot, shRNA, Membrane, Flow Cytometry, CCK-8 Assay

Journal: Cancer Medicine

Article Title: Dihydroartemisinin Sensitizes Lung Cancer Cells to Cisplatin Treatment by Upregulating ZIP14 Expression and Inducing Ferroptosis

doi: 10.1002/cam4.70271

Figure Lengend Snippet: Sequential administration of DDP and DHA has therapeutic differences. (A–C) Western blot was used to detect FTH1 and TFRC expression in A549 and H1975 cells treated with DHA (20 μΜ) and DDP (10 μΜ) alone or in combination at different time points. (D) Flow cytometry was used to detect the expression of ZIP14 in A549 and H1975 cells treated with DHA (20 μM) and DDP (10 μM) at different time points. (E) Cell viability analysis of A549 and H1975 cells with different treatment combinations of DHA (20 μM) and DDP (10 μM) as depicted in (Figure ), mean ± SD, n = 6. (F) Western blot was used to detect the expression of the ferroptosis‐associated proteins in A549 and H1975 cells treated with co‐treatment or sequential treatment of DHA (20 μΜ for A549 or 40 μΜ for H1975) and DDP (10 μΜ). (G) The schematic diagram illustrates drug administration and experimental design for in vivo Lewis lung cancer model. (H) Xenografts of Lewis lung cancer cells were established in C57BL/6 mice and treated with Control (PBS), DHA (20 mg/kg) and DDP (10 mg/kg) simultaneously, or DHA (20 mg/kg) and DDP (10 mg/kg) sequentially. Tumor volume was measured every 3 days and presented as mean ± SEM, n = 5. (I) Representative image of tumor samples in (G) with indicated treatment. (J) Evaluation of MDA in tumor tissue homogenates of mice in (G) with indicated treatment, mean ± SD, n = 3. (K,L) Flow cytometry was used to evaluate the level of ferrous iron and expression of ZIP14 in mice tumor tissues with indicated treatment in (G), mean ± SD, n = 3. (M) The expression of ferroptosis‐related proteins in tumor tissues of mice in (G) with indicated treatment was detected by western blot. * p < 0.05, ** p < 0.01, and *** p < 0.005.

Article Snippet: Antibody against ZIP14 (Affinity, DF14224) was obtained from Affinity (Affinity Biosciences, Jiangsu, China) for flow cytometry and immunofluorescence, while the antibody against ZIP14 (Abclonal, A10413) was obtained from Abclonal (Wuhan, China) for western blot.

Techniques: Western Blot, Expressing, Flow Cytometry, In Vivo, Control

Journal: Cancer Medicine

Article Title: Dihydroartemisinin Sensitizes Lung Cancer Cells to Cisplatin Treatment by Upregulating ZIP14 Expression and Inducing Ferroptosis

doi: 10.1002/cam4.70271

Figure Lengend Snippet: The sequences of (siTFRC) targeting TFRC and (shZIP14) targeting ZIP14.

Article Snippet: Antibody against ZIP14 (Affinity, DF14224) was obtained from Affinity (Affinity Biosciences, Jiangsu, China) for flow cytometry and immunofluorescence, while the antibody against ZIP14 (Abclonal, A10413) was obtained from Abclonal (Wuhan, China) for western blot.

Techniques:

Journal: Cancer Medicine

Article Title: Dihydroartemisinin Sensitizes Lung Cancer Cells to Cisplatin Treatment by Upregulating ZIP14 Expression and Inducing Ferroptosis

doi: 10.1002/cam4.70271

Figure Lengend Snippet: Decreased expression of ZIP14 contributed to the tolerance of DHA&DDP‐induced ferroptosis. (A) The qPCR analysis of ZIP14, ZIP8, and DMT1 levels in A549 cells treated with DHA (20 μM) and DDP (10 μM) alone or in combination for 48 h, mean ± SD, n = 3. (B) Immunofluorescence imaging of ZIP14 (red) and DAPI (blue) under mono or combined treatment of DHA (20 μΜ) and DDP (10 μΜ) in A549 cells for 48 h. Scale bars: 100 μm. (C) Western blot was used to detect the expression of ZIP14 proteins in A549 and H1975 cells treated with DHA (20 μΜ for A549 or 40 μΜ for H1975) and DDP (10 μΜ) alone or in combination for 48 h. (D) The level of ZIP14 mRNA was measured by qPCR after RNA interference using shRNA in A549 cells, mean ± SD, n = 3. (E) Western blot was used to detect the expression ZIP14 proteins in A549 cells treated with shZIP14. (F) Membrane‐associated ZIP14 protein levels in shNC or shZIP14 A549 cells were measured using flow cytometry. (G) The viability of shNC or shZIP14 A549 cells treated with DHA (20 μM) and DDP (10 μM) for 48 h by CCK‐8 assay, mean ± SD, n = 3. (H) Western blot was used to detect the expression of the ferroptosis‐associated proteins in shNC or shZIP14 A549 cells treated with DHA (20 μM) and DDP (10 μM) for 48 h. (I) Flow cytometry was used to evaluate changes in ROS (left) and ferrous iron (right) levels in shNC or shZIP14 A549 cells treated with DHA (20 μM) and DDP (10 μM) for 48 h. *** p < 0.001.

Article Snippet: Antibody against ZIP14 (Affinity, DF14224) was obtained from Affinity (Affinity Biosciences, Jiangsu, China) for flow cytometry and immunofluorescence, while the antibody against ZIP14 (Abclonal, A10413) was obtained from Abclonal (Wuhan, China) for western blot.

Techniques: Expressing, Immunofluorescence, Imaging, Western Blot, shRNA, Membrane, Flow Cytometry, CCK-8 Assay

Journal: Cancer Medicine

Article Title: Dihydroartemisinin Sensitizes Lung Cancer Cells to Cisplatin Treatment by Upregulating ZIP14 Expression and Inducing Ferroptosis

doi: 10.1002/cam4.70271

Figure Lengend Snippet: Sequential administration of DDP and DHA has therapeutic differences. (A–C) Western blot was used to detect FTH1 and TFRC expression in A549 and H1975 cells treated with DHA (20 μΜ) and DDP (10 μΜ) alone or in combination at different time points. (D) Flow cytometry was used to detect the expression of ZIP14 in A549 and H1975 cells treated with DHA (20 μM) and DDP (10 μM) at different time points. (E) Cell viability analysis of A549 and H1975 cells with different treatment combinations of DHA (20 μM) and DDP (10 μM) as depicted in (Figure ), mean ± SD, n = 6. (F) Western blot was used to detect the expression of the ferroptosis‐associated proteins in A549 and H1975 cells treated with co‐treatment or sequential treatment of DHA (20 μΜ for A549 or 40 μΜ for H1975) and DDP (10 μΜ). (G) The schematic diagram illustrates drug administration and experimental design for in vivo Lewis lung cancer model. (H) Xenografts of Lewis lung cancer cells were established in C57BL/6 mice and treated with Control (PBS), DHA (20 mg/kg) and DDP (10 mg/kg) simultaneously, or DHA (20 mg/kg) and DDP (10 mg/kg) sequentially. Tumor volume was measured every 3 days and presented as mean ± SEM, n = 5. (I) Representative image of tumor samples in (G) with indicated treatment. (J) Evaluation of MDA in tumor tissue homogenates of mice in (G) with indicated treatment, mean ± SD, n = 3. (K,L) Flow cytometry was used to evaluate the level of ferrous iron and expression of ZIP14 in mice tumor tissues with indicated treatment in (G), mean ± SD, n = 3. (M) The expression of ferroptosis‐related proteins in tumor tissues of mice in (G) with indicated treatment was detected by western blot. * p < 0.05, ** p < 0.01, and *** p < 0.005.

Article Snippet: Antibody against ZIP14 (Affinity, DF14224) was obtained from Affinity (Affinity Biosciences, Jiangsu, China) for flow cytometry and immunofluorescence, while the antibody against ZIP14 (Abclonal, A10413) was obtained from Abclonal (Wuhan, China) for western blot.

Techniques: Western Blot, Expressing, Flow Cytometry, In Vivo, Control

Journal: Cancer Medicine

Article Title: Dihydroartemisinin Sensitizes Lung Cancer Cells to Cisplatin Treatment by Upregulating ZIP14 Expression and Inducing Ferroptosis

doi: 10.1002/cam4.70271

Figure Lengend Snippet: The sequences of (siTFRC) targeting TFRC and (shZIP14) targeting ZIP14.

Article Snippet: Antibody against ZIP14 (Affinity, DF14224) was obtained from Affinity (Affinity Biosciences, Jiangsu, China) for flow cytometry and immunofluorescence, while the antibody against ZIP14 (Abclonal, A10413) was obtained from Abclonal (Wuhan, China) for western blot.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Identification and verification of a novel signature that combines cuproptosis-related genes with ferroptosis-related genes in osteoarthritis using bioinformatics analysis and experimental validation

doi: 10.1186/s13075-024-03328-3

Figure Lengend Snippet: Experimental validation of four biomarkers. a Representative immunofluorescence staining images of the four biomarker proteins (p21, FZD7, GABARAPL2, and ZIP14) in the normal and OA groups, with nuclei stained blue with 4’,6-diamidino-2-phenylindole. Scale bar = 25 µm. b Semi-quantitative analysis of mean fluorescence intensity of the four biomarker proteins in the normal and OA groups ( n = 6). (c, d) Representative western blotting and statistical comparisons of the four biomarker proteins in the normal and OA groups ( n = 6). * p < 0.05, ** p < 0.01, all by independent samples t-test

Article Snippet: The antibodies used for immunofluorescence and western blot in this study were as follows: rabbit anti-FZD7 (Cat. #: DF8657, 1:1,000; AFFBIOTECH, USA), rabbit anti-SLC39A14 (ZIP14) (Cat. #: 26540-1-AP, 1:1,000, Proteintech, Rosemont, IL, USA), rabbit anti-CDKN1A (p21) (Cat. #: 2947T, 1:1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GABARAPL2 (Cat. #: 14256T, 1:1,000, Cell Signaling Technology), anti-GAPDH (Cat. #: 60004 -1-Ig, 1:1,000, Proteintech, USA), and species-matched HRP-conjugated secondary antibody (Cat. #: SA00001-1, 1:1,000; Proteintech, USA).

Techniques: Biomarker Discovery, Immunofluorescence, Staining, Fluorescence, Western Blot