FIGURE 3.

High glucose increased PICALM, AP2A1, and CHC expression through ROS‐stimulated Sp1 nuclear translocation. (a) The SK‐N‐MC cells were treated with distilled water (DW) or 25 mM high glucose in a time response. Intracellular ROS was measured by DCF‐DA staining and analysed by flow cytometry. n = 5 from independent experiments. (b,c) The cells were incubated with NAC (4 mM) for 30 min prior to high glucose treatment (25 mM) for 6 h. (b) Sp1, β‐tubulin, and Lamin A/C in cytosolic and nuclear fraction samples were detected by western blot. n = 5 from independent experiments. (c) The cells were immunostained with Sp1‐specific antibody and counterstained with DAPI. Scale bars, 8 μm (magnification, ×1,000). n = 5 from independent experiments. (d) The cells were pretreated with Mithramycin A (25 nM) for 30 min before high glucose treatment (25 mM) for 12 h. mRNA expression levels of PICALM, AP2A1, and CHC were analysed by quantitative real‐time PCR. n = 5 from independent experiments with two technical replicates each. Logarithmic transformations were performed for homogeneity of the sample variance. (e) The cells were incubated with Mithramycin A (25 nM) for 30 min prior to high glucose treatment (25 mM) for 24 h. PICALM, AP2A1, CHC, and β‐actin were subjected to western blot. n = 5 from independent experiments. *P < .05, significantly different from control, ^# P < .05, significantly different from high glucose. (f) The hippocampal samples were obtained from ZLC and ZDF rats. PICALM, AP2A1, CHC, and β‐actin were detected by western blot. n = 5 in each group. *P < .05, significantly different from ZLC rats. (g) The hippocampal samples were obtained from vehicle‐ and STZ‐treated mice. PICALM, AP2A1, CHC, and β‐actin were detected by western blot. n = 5 in each group. *P < .05, significantly different from vehicle‐treated mice. Quantitative data are presented as a mean ± SEM. All blots and immunofluorescence images are representative