Knockdown of XBP1 down-regulates anti-oxidant genes in human RPE cells.

<p>Human RPE (ARPE-19) cells were transfected with XBP1 siRNA or control siRNA for 48 h. <b>A</b>). mRNA expression of antioxidant gene catalase, SOD2, SOD1, Nrf2, and GSH synthase was measured by real-time RT-PCR. Data were expressed as mean ± SD (n = 3 independent experiments). <b>B</b>). Protein levels of spliced XBP1and SOD2 were determined by Western blot analysis and semi-quantified by densitometry. <b>C</b>). Intracellular superoxide production was detected by Dihydroethidium (DHE) (upper panel). Mitochondrial superoxide level was analyzed by MitoSOX™ Red assay (lower panel). Representative images from 3 independent experiments are shown. <b>D</b>). Intracellular ROS generation was determined by DCF. The fluorescence density was quantified by using a fluorescence plate reader with wavelength of 485/535 nm (mean ± SD, n = 3). <b>E</b>). Cell viability was determined by MTT assay. The numbers of viable cells are expressed as % of control, averaged from 3 independent experiments (mean ± SD). <b>F</b>). Apoptosis was detected by Annexin V staining in ARPE-19 cells transfected with XBP1 siRNA or control siRNA. * <i>P</i><0.05, ** <i>P</i><0.01 vs. control siRNA. <b>G</b>). Transfected cells were exposed to 4-HNE (50 µM) for 24 h, apoptosis was detected by TUNEL assay. Left panels show representative images of TUNEL staining (red). Nuclei were stained with DAPI (blue). Right panel shows the quantitative results of apoptotic cells. * <i>P</i><0.05 vs. control siRNA; ‡ <i>P</i><0.01 vs. control siRNA+4-HNE.