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. 2011 Dec 12;61(1):208–216. doi: 10.2337/db11-1024

FIG. 3.

FIG. 3.

Egr1 mediates glucose-induced overexpression of heparanase in the kidney. A and B: Effects of glucose on Egr1 and heparanase expression in vitro. HEK-293 cells were incubated (1.5 h) in triplicates in the absence or presence of increasing concentrations of glucose (1, 2.5, and 4.5 g/L). A: Egr1 expression was assessed by immunoblotting of nuclear extracts (top) and qRT-PCR determination of mRNA levels (bottom). B: Heparanase mRNA expression was assessed by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001. C and D: Egr1 induction in the kidneys of diabetic mice correlates with overexpression of heparanase. C: Induction of Egr1 in the kidneys of diabetic mice. Kidney tissue was harvested from nondiabetic and diabetic mice 10 weeks after induction of diabetes by STZ, lysed, and analyzed for Egr1 expression by qRT-PCR determination of mRNA levels. Error bars represent ± SE (n = 4 mice for each group). *P < 0.05. D: Heparanase expression in nondiabetic and diabetic kidney lysates was assessed by qRT-PCR (left), immunostaining (left, inset), enzymatic activity assay (right), and immunoblotting (right, inset). E: Increased recruitment of Egr1 to the heparanase promoter in the presence of high glucose. After cross-linking of proteins to DNA, chromatin was isolated from HEK-293 cells incubated (1.5 h) in the presence of increasing concentrations of glucose (0, 1, and 4.5 g/L), sonicated into fragments of average length ≤ 500 base pairs and immunoprecipitated with antibodies against Egr1 (top), or an unrelated protein LAMP1 (middle), as described in research design and methods. The final DNA extractions were amplified using primer set that covers functional Egr1 binding site in the heparanase promoter (left), or primer set specific to unrelated L-19 gene sequence, used as control (right). Samples were equilibrated for DNA loading amounts using primers specific to the heparanase promoter and DNA that was PCR amplified from chromatin preparations before immunoprecipitation (bottom). The results are representative of three independent experiments. (A high-quality color representation of this figure is available in the online issue.)