FIGURE 6.

Knockdown of Nrf2 increases autophagy and cell cycle arrest but not apoptosis by MitoQ. A, conditions for Nrf2 knockdown with siRNA were optimized in MDA-MB-231 cells. Increasing concentrations (at 72 h) and incubation times (with 100 nm) siRNA oligonucleotides were tested. The bands were quantitated using densitometric scanning and normalized to α-tubulin. The relative levels of Nrf2 are indicated below the images. Data shown is representative of three independent experiments. B, increased levels of the autophagosome-associated LC3-II subunit were used as a measure of autophagy 24 h after siRNA. MitoQ (1 μm) was tested at 24 and 72 h. Non-coding siRNA oligonucleotides were used as control siRNA. Data from two individual experiments (Expt.) are shown. C, percentage of cells with autophagosomes after Nrf2 or control siRNA transfection followed by MitoQ treatment were counted as in Fig. 3D. *, statistical significance compared with control siRNA cells. D, cell cycle profiles of Nrf2 or control siRNA cells exposed to MitoQ were analyzed by flow cytometry following staining with PI. The percentage of cells in each cell cycle phase was quantified and is provided in each panel. Right, apoptosis was measured in live Nrf2 siRNA MDA-MB-231 cells treated with 1 μm MitoQ for 72 h. The percentage of cells that were annexin V-positive was used as a measure of apoptosis. Error bars, S.D.