1. Nrf2 negatively regulated TGF-β1 and FN in HRMCs
Nrf2 negatively regulated TGF-β1 and FN in HRMCs. HRMCs growing were transfected with plasmids for TGF-β1–promoter-firefly luciferase and renilla luciferase (internal control), along with different amounts of an expression vector for Nrf2.
Nrf2 negatively regulated TGF-β1 and FN in HRMCs. HRMCs growing were transfected with plasmids for TGF-β1–promoter-firefly luciferase and renilla luciferase (internal control), along with different amounts of an expression vector for Nrf2. At 48 h post-transfection, both firefly and renilla luciferase activities were measured (A, upper panel). An aliquot of cell lysates was used for immunoblot analysis (A, lower panel). HRMCs were transfected with plasmids for TGF-β1–promoter-firefly luciferase and renilla luciferase. Cells were then treated with tert-butylhydroquinone (tBHQ) for 16 h before the measurement of luciferase at 48 h post-transfection (B, upper panel). An aliquot of cell lysates was used for immunoblot analysis (B, lower panel). HRMCs were transfected with control- or Nrf2-siRNA. Then 24 h later, cells were starved before incubation with low- or high-glucose medium for an additional 24 h. Total mRNAs were extracted, and qRT-PCR was performed to measure the mRNA level of TGF-β1 (C). Another parallel set of cells was collected in lysis buffer for immunoblot analysis with antibodies against Nrf2, aldose reductase (AR), γGCS, NQO1, FN, and GAPDH (D, upper panel). The intensity of the bands was calculated and quantified (D, lower panel). All the experiments were repeated three times and data represent means ± SD.
Tao Jiang et al. Diabetes 2010;59:
©2010 by American Diabetes Association