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. 2020 Apr 20;27(9):2668–2680. doi: 10.1038/s41418-020-0535-y

Fig. 3. Elevated Nox4 stimulates NRF2-related responses to reduce epidermal ROS.

Fig. 3

a Representative immunoblots of Nox4 and NRF2 in epidermis from FSPCre-Pparb/dex4 and Pparb/dfl/fl mice. Densitometric quantification plots are shown (top panel). β-tubulin served as a housekeeping protein from the same samples. b Extracellular H2O2 levels in FSPCre-Pparb/dex4 and Pparb/dfl/fl keratinocytes. Extracellular H2O2 was detected by the Amplex Red Assay and normalized to cell number. c Histogram shows the mean intracellular ROS (CellRox fluorescence readings) in cytokeratin-positive cells from the skin of FSPCre-Pparb/dex4 (red) and Pparb/dfl/fl skin (gray). d Fold change in ARE-dependent luciferase activities in keratinocytes cocultured with primary fibroblasts from FSPCre-Pparb/dex4 or Pparb/dfl/fl mice. For the reporter assay, firefly luciferase activity was normalized to Renilla luciferase activity. e Relative mRNA levels of the indicated antioxidant genes in the epidermis from FSPCre-Pparb/dex4 and Pparb/dfl/fl mice. Ribosomal protein L27 was used as a housekeeping gene. f Relative mRNA level of NRF2 in human primary keratinocytes treated with vehicle and kinase inhibitors against B-Raf and MEK1/2 at 4 h post H2O2 stimulation. Ribosomal protein L27 was used as a housekeeping gene. g Representative immunoblots of total and phosphorylated B-Raf, MEK1/2, and ERK1/2 in human keratinocytes treated with H2O2 (left panel). A densitometric quantification plot is shown (right panel). Total B-Raf, MEK1/2, and ERK1/2 as cognate normalizing proteins were from the same samples. h Representative immunoblots of Nox4 and NRF2 in human keratinocytes (Kers) cocultured with either dermal human fibroblasts knockdown of Pparb/d (FsiPparb/d) or scrambled siRNA (Fscrambled) (bottom panel). β-tubulin served as housekeeping protein and was from the same samples. A densitometric quantification plot is shown (top panel). Data are represented as the mean ± s.d. For a and d–h, n = 3 independent experiments. For b, c, n = 5 independent experiments. *p < 0.05, **p < 0.01 and n.s. denotes non-significant.