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. 2008 Jul;19(7):2984–2994. doi: 10.1091/mbc.E08-02-0138

Figure 1.

Figure 1.

HT29 cell ROS generation is dependent on the Nox1 pathway. (A) The Nox1 siRNA#3 was the most effective in specifically knocking down the level of Nox1 mRNA in a dose-dependent manner. HT29 cells were transfected with the indicated amounts of three different Nox1-specific siRNAs or with control siRNA, and total RNA was extracted after 72 h as in Materials and Methods. RT-PCR was performed using Nox1-specific primers (top) or actin-specific primers (middle) and PCR products were run on a 1% agarose gel. Bottom, total RNA from each extraction was run on 1% agarose, showing no difference in the amount of the total RNA between different experimental conditions. (B) The transfection of Nox1-specific siRNA #3 results in the strongest dose-dependent reduction of cellular ROS production. HT29 cells were transfected (as indicated) with different amounts of three different Nox1-specific siRNAs or with control siRNA, and after 72 h ROS generation was measured by CL-assay as described in Materials and Methods. One representative experiment from three separate experiments is shown, and results are given as mean of triplicates ± SD. (C) The inhibition of ROS generation caused by Nox1 siRNA#3 in HT29 cells is rescued by the overexpression of mNox1. HT29 cells were transfected with control siRNA or Nox1 siRNA#3 (20 nM) as described in Materials and Methods. After 48 h, Nox1 siRNA-transfected cells were transfected again with expression plasmid for mNox1 or with empty vector by Lipofectamine 2000. Twenty-four hours later ROS generation was measured. One representative experiment from three separate experiments is shown, and results are given as mean of triplicates ± SD. (D) Treatment of HT29 cells with 10 μM DPI, a flavoenzyme inhibitor of NADPH oxidases, blocks both the increase in ROS production due to the transfection of Nox1, NoxA1, and NoxO1, and it reduces the amount of cellular ROS in mock-transfected HT29 cells. HT29 cells were transfected as indicated with empty vector or with the expression vector for Nox1, NoxO1, and NoxA1. After 24 h, cells were treated with 10 μM DPI or DMSO, and ROS production was monitored by CL assay. One representative experiment from three separate experiments is shown, and results are given as mean of triplicates ± SD.