Representative specimens from patients with NPC showed weak (a) and strong (b) cell membrane immunostaining for ZIP4 at different NPC stages (Scale bar = 101 μm). The results of the Kaplan–Meier survival analysis indicate that patients with high ZIP4 expression present a shorter overall survival (OS) (c), recurrence-free survival (RFS) (d), and distant metastasis-free survival (DMFS) (e). The differences in OS, RFS, and DMFS between the ZIP4 low and high expression levels were significant, low/high = 25/74 (P < 0.001)

a Relative protein and mRNA level of ZIP4 were detected in stable LVRH, Sh-ZIP4, Control, and ZIP4 cell lines. b The cell migration rate between LVRH C666-1 and Sh-ZIP4 C666-1 cells was compared by the wound-healing assay. Microscopic observation was recorded at 0 and 24 h after scratching the cell layer (Scale bar = 50 μm). c The invasive and migration properties of the cells were analyzed by using a matrigel-coated Boyden chamber. Migrated cells were plotted as the average number of cells per field from three different experiments (Scale bar = 50 μm). d The expression levels of EMT markers, E-cadherin (E-cad), vimentin (Vim), and FSP-1 were examined by immunofluorescence staining (Scale bar = 75 μm). e E-cad, vimentin (Vim), and FSP-1 expression were examined in control and ZIP4 C666-1 cells by western blot. The experiments were performed in triplicate. The data are represented as means ± SD from three independent experiments. *P < 0.05

a, b Red fluorescence-labeled tumor cells were injected into the perivitelline space of 48 h postfertilization embryos and tumor cell invasion, dissemination, and metastasis were detected using confocal microscopy at day 3 post injection. Yellow arrows indicate primary tumors. White arrows indicate disseminated tumor foci (Scale bar = 100 μm). c Quantification of disseminated tumor foci (n = 40 per group), P < 0.01. d Representative images of the mouse lungs. e The average number of tumor metastatic nodules in the lungs of mice xenografted with C666-1, LVRH C666-1 cells, and Sh-ZIP4 C666-1 cells is provided (N = 7; P < 0.01). f Representative views of lung tissue sections from each group are shown (Scale bar = 50 μm). The experiments were performed in triplicate. The data are represented as means ± SD from three independent experiments

a KEGG pathway analysis of the discrepantly expressed genes in Sh-ZIP4 compared with control LVRH C666-1 cells. b Phospho-antibody array. See Supplementary Fig. 1 for full results and additional annotations. c The western blot assay of the selected proteins for the determination of phosphorylation antibody array. d Effect of PI3K inhibitors, LY294002, and Wortmannin on EMT markers. The experiments were performed in triplicate. The data are represented as means ± SD from three independent experiments. *P < 0.05

a Cell growth in low and high concentration of zinc. b Inhibition of cell proliferation following exposure to various doses of Co⁶⁰ was evaluated by the MTT assay (P < 0.01). c Images and quantification of the number of colonies formed from cells treated with 0, 2, 4, 6, and 8 Gy (P < 0.05). d Flow cytometry analysis of apoptosis using annexin V and propidium iodide in cells treated with various doses of radiation (0, 2, 4, 6, and 8 Gy). e Flow cytometric analysis of the cell cycle in cells treated with various doses of radiation. f Protein expression levels of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 in cells treated with a 6 Gy dose of radiation. Experiments were repeated three times. The data are represented as means ± SD from three independent experiments. *P < 0.05

a, b Sh-ZIP4 and LVRH C666-1 cells (1 × 10⁷) were subcutaneously inoculated into the right hind limb of nude mice (N = 7 per group). When the size of the tumors reached about 50 mm³, tumors were irradiated at 5 Gy per day for 4 days (on day 1, 3, 6, and 8). Tumor size was measured and weight was recorded. c TUNEL staining of C666-1 xenografts (Scale bar = 50 μm) (P < 0.01). d PCNA IHC staining of C666-1 xenografts (P < 0.01). e Overexpression of ZIP4 induces the EMT process through the PI3K/Akt pathway and further promotes migration and invasion of NPC cells. Meanwhile, ZIP4 enhanced radiation-induced apoptosis and growth inhibition of human C666-1 cells. These findings support our clinical observations, indicating that ZIP4 elevation is associated with clinical T classification, N classification, and clinical stage, and leads to adverse survival outcomes of NPC