FIGURE 7.

Effects of the addition of excessive amounts of zinc and the removal of zinc on the differentiation of HDK1. a, gene expression analysis of IVL on day 4 of differentiation induction. The expression of IVL was restored by adding 50 μm of ZnCl₂. b, images of HDK1 in which ZIP2 was knocked down by siRNA. On day 4 of differentiation, HDK1 did not exhibit a cobblestone morphology by knockdown of ZIP2. However, HDK1 exhibited a cobblestone morphology by addition 50 μm of ZnCl₂. c, quantitative analysis of zinc in the culture medium by atomic absorption spectrophotometry. The zinc level in the culture medium was depleted by treatment with Chelex-100. d, gene expression analysis of IVL and ZIP2 on day 4 of differentiation induction. The expression of ZIP2 was enhanced notably by removing zinc in the culture medium by treatment with Chelex-100. e and f, intracellular zinc levels in HDK1 (control and Chelex-100) on day 4 of differentiation. The ratio of FluoZin-3 ^high cells was not changed by treatment with Chelex-100. Scale bar = 200 μm. Data are mean ± S.D. of three experiments. *, p < 0.05; **, p < 0.01; significantly different from the control.