Fig. 3. Constitutive NRF2 nuclear localization increases resistance to oxidative stress.
a Chicken (CPF) and Neoaves (Dumetella carolinensis) primary fibroblasts (NPF) were used to evaluate KEAP1 regulation of NRF2 in vitro. “Gray Catbird” [CC BY 2.0] Andy Reago & Chrissy McClarren. https://flickr.com/photos/80270393@N06/17166164877. No changes were made. b CDDO-Im is a chemical modulator of KEAP1 that induces release of NRF2 and consequent gene transcription at nuclear antioxidant response elements (AREs; “Methods”). c Immunocytochemistry and confocal microscopy (scale bar (white) for all micrographs is 20 µm) of cells exposed to vehicle (DMSO) or 50 nM of CDDO-Im. NRF2 immunofluorescence in CPF and NPF cells. This experiment was repeated independently N = 5 times, with similar results. d Luciferase assays of vehicle- and CDDO-Im-treated CPF cells [N = 10 biologically independent cells; ***p = 5.07 × 10−12, one-sided t test] and NPF cells [N = 12 biologically independent cells]. e Multiple NRF2 target genes are significantly upregulated in Chicken cells upon CDDO-Im treatment [N = 2 biologically independent cells] relative to vehicle [N = 3 biologically independent cells]. These same target genes are constitutively expressed at high levels in Neoaves cells and show no response to CDDO-Im [N = 3]. p Values are noted above each comparison. One-sided t test. f Significantly decreased oxidative stress in NPF cells relative to CPF cells when challenged with various concentrations of tert-butyl hydroperoxide (tBh) [N = 6 biologically independent cells, except for at 500 µM tBh where N = 5 biologically independent cells]. 0 μM, *p = 0.045; 100 μM, **p = 0.002; 500 μM, ***p = 1.54 × 10−5. Two-sided t test. g Human cells (HEK293T) with Cas9-induced KEAP1 loss of function display significantly decreased oxidative stress relative to wild type when challenged with 500 μM tBh [N = 5 biologically independent cells]. Two-sided t test. *(p < 0.05); **(p < 0.01); ***(p < 0.001). This oxidative stress reduction is strikingly similar to that between NPF and CPF cells. h Oxidative stress is increased in NPF cells transfected with Chicken KEAP1 (C) relative to those transfected with Neoaves KEAP1 (N; Taeniopygia guttata) or mock transfected (−) and exposed to various concentrations of tBh [100 μM, N = 2 biologically independent cells; 0 μM, N = 3 biologically independent cells]. Mock vs. C, **p = 0.006; Mock vs. N, p = 0.14; C vs. N, *p = 0.014; two-sided t test. Fluorescence Ex/Em: 492–495/517–527 nm. Source data are provided as a Source data file. All data are presented as mean values. All error bars represent standard error.