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. 2016 May 2;213(5):859–875. doi: 10.1084/jem.20151208

Figure 5.

Figure 5.

Nrf2 promotes gankyrin transcription. (A) Schematic representation of gankyrin promoters in humans (Homo species, hGank), pigs (Sus scrofa, sGank), mice (Mus musculus, mGank), rats (Rattus norvegicus, rGank), and chimpanzees (Pan troglodytes, pGank). Different symbols represent Nrf2 binding sites (ARE) in different species. TSS, transcriptional start site. (B) ARE regions and adjacent sequences in the gankyrin promoter. (C) SMMC7721 and MHCC-LM3 cells were fixed and sheered; cross-linked chromatin was prepared as described in the Materials and methods. The chromatin was precipitated using control (IgG) or Nrf2-specific antibodies (Nrf2). PCR analysis was performed using primers for ARE1, ARE2, and ARE3. The data shown are representative of three independent experiments. (D) Oxidative stress increases the binding of Nrf2 to the AREs of the gankyrin promoter. SMMC7721 and MHCCLM3 cells were treated with PBS or 0.5 mM H2O2 for 5 h, and chromatin immunoprecipitation was performed using Nrf2-specific antibodies. DNA isolated from the precipitated materials was analyzed using qPCR with the indicated primers. The ARE-specific signals from Nrf2-precipitated DNA were normalized to those from IgG-precipitated DNA. The data shown are means ± SEM of triplicate wells. (E and F) qRT-PCR analysis was performed for gankyrin and target genes of Nrf2 in PLC/RPF/5-con and PLC/RPF/5-siNrf2 or SMMC7721-con and SMMC7721-siNrf2 cells. Data represent the mean ± SEM of triplicates from an experiment that was repeated a total of three times with similar results. *, P < 0.05; **, P < 0.01. (G) Western blot analysis of gankyrin and NQO1 in SMMC7721-con, SMMC7721-siNrf2, PLC/RPF/5-con, and PLC/RPF/5-siNrf2 cells. Representative results from three experiments are shown. (H) Effects of sulforaphane and tBHQ on gankyrin expression in HCC cells. SMMC7721 cells were treated with 0 to 5 µM sulforaphane and tBHQ for 12 h, and the cells were then lysed and subjected to Western blot analysis. Representative results from three experiments are shown.