Fig. 1.
The ZIP6/ZIP10 heteromer is increased in mitosis. a Non-permeabilised Mitotic MCF-7 cells (arrow) have increased staining for ZIP6 (red, ZIP6-Y antibody) and ZIP10 (green, ZIP10B antibody) on the plasma membrane in contrast to the non-mitotic cells. Nuclei stained blue with DAPI. b, c FACS cell cycle analysis confirms the increase compared to control in the G2/M population in nocodazole-treated cells (b) as well as the increase in non-adherent cells collected after mitotic shake-off (c), compared to the adherent cells from the same dishes. Results of three independent experiments are demonstrated as mean ± SD. Statistical significance is determined by Student’s t test.***p < 0.001. d MCF-7 cells treated with nocodazole for 20 h have increased mitosis as judged by mitotic marker pS10HistoneH3 as shown from a representative blot of more than three experiments. ZIP6 is significantly increased in mitotic cells and the active N-terminal-cleaved form of ZIP6 as a 68 kDa band is recognised by both ZIP6 antibodies (ZIP6-Y recognises N-terminus and ZIP6-SC recognises the cytoplasmic loop between TM3-4). ZIP10 undergoes cleavage in mitosis; Using the N-terminal antibody ZIP10B the full length protein (105 kDa) decreased in the nocodazole-treated population and the smaller cleaved fragment increased significantly (45 kDa). Using the cytoplasmic loop antibody ZIP10S, an increase in the 60 kDa band was observed in mitosis which corresponded to the remainder of ZIP10 after removal of some of the N-terminus. Statistics (student t test) were compared to control. e FACS cell cycle analysis of transfected cells, divided into adherent and non-adherent populations after mitotic shake off showing that transfection with ZIP6 or ZIP10 increased the cells in G2/M whether from the adherent on nonadherent populations. Results of at least three independent experiments are shown as mean ± SD Statistical significance comparing results to control samples was performed using ANOVA with Dunnett post-hoc and is shown as ***(p < 0.01) or ***(p < 0.001). f Proximity ligation assay (PLA) using ZIP6-Y and ZIP10B antibodies in nocodazole-treated MCF-7 cells, produces significantly increased dots (red) in mitotic cells (white arrows) compared to non-mitotic cells. Nuclei stained with DAPI (blue). Representative picture of quantitative measurements from at least 6 images of 25 stacks taken 0.3 μm apart from 3 independent experiments are demonstrated as mean ± standard error. Statistical significance was compared between mitotic and non − mitotic cells using ANOVA with Dunnett post-hoc and is shown as ***p < 0.001. Scale bar, 25 μm