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. 2019 Apr 23;30(17):1983–1998. doi: 10.1089/ars.2017.7486

FIG. 1.

FIG. 1.

The effects of high glucose on PP2Cδ expression, p53 activity and cell proliferation, migration, and invasion. MCF-7 (A) and normal MCF-12A (C) cells were treated with high glucose (22.5 mM) for 0–48 h with a change of fresh medium every 24 h. In a dose–response study, MCF-7 (B) and MCF-12A (D) cells were treated with different concentrations of glucose as indicated for 24 h. The cells were then lysed. Nuclear fractions were prepared and underwent SDS-PAGE followed by Western blotting with various primary antibodies as shown, then were visualized by the enzyme-linked chemiluminescence system. The bar graphs above are densitometry analyses of the bands. The total amount of TBP is shown for equal loading. Data presented are mean ± SD from three independent experiments, with nontreated controls set as 1. Uncropped blots are shown in Supplementary Figure S1. *p < 0.05 versus control. (E) Induction of PP2Cδ mRNA expression in MCF-7 cells treated with HG analyzed by qRT-PCR. All mRNA were normalized to PUM1 and presented as fold (mean ± SD) over control based on three experiments. *p < 0.05 versus control. (F) MCF-7 cells were transiently transfected with 0.5 μg of pG13-LUC reporter plasmid. About 6 h after transfection, cells were treated HG alone or pretreated with UV for 0.5 h followed by HG treatment. Luciferase activity was determined from the transfected cell extracts. Values (mean ± SD) are expressed as fold over untreated control. *p < 0.05, #p < 0.05 versus their corresponding control groups. (G) MTT cell proliferation assays illustrating the effects of HG on fold cell proliferation compared with a NG condition in MCF-7 cells. *p < 0.05 versus control (n = 3). (H) Wound-healing assay of MCF-7 cells treated with HG. MCF-7 cells were seeded in migration chambers and treated with 22.5 mM HG. Images of the migration area were photographed 0, 24, and 48 h after removal of the migration chamber under a phase contrast microscope. The percentage of the wound closed was quantified from three independent replicates and is expressed as mean ± SD. (I) In vitro invasion assay performed on MCF-7 cells that were treated with HG for 24 and 48 h. Each column (right) represents the mean (±SD) results of three independent experiments. *p < 0.05, versus their corresponding control groups. HG, high glucose; NG, normal glucose; PP2C, type 2C protein phosphatase; PUM1, pumilio RNA-binding family member 1; TBP, TATA-binding protein. Color images are available online.