Fig. 5

Nrf2 regulates the eEPCs-to-macrophage conversion in response to chronic TNFα stimulation. A eEPCs were exposed to 50 ng/ml TNFα for the indicated time. Total cytoplasmic and nuclear proteins were extracted for Western blot using an anti-Nrf2. Tubulin and Histone are shown as loading control, respectively. # non-specific bands. B Alternatively, cells were subjected to immunofluorescence analysis of Nrf2 nuclear translocation. Nrf2 was probed with a primary anti-Nrf2 antibody and visualized with an Alexa Fluor 594-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). C–F eEPCs were transfected with a siRNA targeting chicken Keap1 or a negative control (NC) siRNA for 48–72 h. C Evaluation of knockdown efficiency of Keap1. Total RNAs and whole-cell lysates were subjected to qPCR and Western blot analysis, respectively, for determination of Keap1 mRNA (left panel, n = 3) and protein levels (right panel). D Evaluation of Nrf2 activation after siRNA silencing of Keap1. Total RNAs and whole-cell lysates were subjected to Western blot and qPCR analysis, respectively, for determination of Nrf2 protein levels (left panel) and relative mRNA level of Nrf2 target genes Nqo-1 (NAD(P)H:quinone oxidoreductase 1) (right panel, n = 3). E–F Effect of Nrf2 activation on the mRNA expression of stem/progenitor cell marker CD133 and macrophage marker MRC1 (n = 3). G HEK-293T cells were co-transfected with the chNrf2 expression vector pEGFP-C3-Nrf2 (denoted in μg of DNA per well of a 12-well plate) and the plasmid constructs containing a firefly luciferase gene under the control of the MRC1 promoter along with the pRL-TK Renilla. Empty pEGFP-C3 was used as control. Cells were incubated with or without TNFα (50 ng/ml) for 72 h and processed for measurement of luciferase activity (n = 3). The data are representative of at least 2 separate experiments with similar results. Data were expressed as median ± 95% confidence interval. *P < 0.05