Fig. 4.

NOX complexes catalyze PDGF-evoked SHP2 oxidation. a Serum-starved Swiss 3T3 cells expressing EGFP or cytoplasmic catalase were stimulated with PDGF-BB (50 ng ml⁻¹) or H₂O₂ (1 mM) for 10 min and subjected to dimedone-PLA (gray). Representative images are shown for each condition from one of two independent experiments. b Serum-starved Swiss 3T3 cells were left untreated or were pre-treated with DPI (2.5 μM) (top) or imipramine-blue (6 μM) (bottom) for 5 min, stimulated with PDGF-BB (50 ng ml⁻¹) or H₂O₂ (1 mM) for 10 min and subjected to dimedone-PLA (gray). Representative images are shown for each condition from one of two independent experiments. c Serum-starved primary murine dermal fibroblasts (MDFs) from wild type (WT) or Nox1, 2, 4 triple-KO (TKO) mice were stimulated with PDGF-BB (50 ng ml⁻¹) or H₂O₂ (1 mM) for 10 min and subjected to dimedone-PLA (gray). Representative images are shown for each condition from one of three independent experiments. a–c The graphs show the average number of PLA signals per cell (n = 6 images for each condition, 5–15 cells in an image), relative to control cells (normalized to 1). Error bars represent SD. ***P < 0.0001, ns not significant, ANOVA with Bonferroni/Dunn’s post-hoc test. Scale bar: 50 μm. d Serum-starved Swiss 3T3 cells were stimulated with PDGF-BB (50 ng ml⁻¹) for 10 min, and fixed in the presence of dimedone. Representative co-staining of ox-SHP2, detected by dimedone-PLA (magenta), and the indicated NOX components (green) are shown from one of three independent experiments (top). Nuclei were stained with DAPI (blue). Higher magnification images of the boxed regions are shown. Scale bar: 10 μm. Median distances of centers of mass (CM) between punctate signals of ox-SHP2 and the nearest indicated NOX protein signal in cells were obtained by object-based image analysis. Box-whisker plots show median distances at 10 min after PDGF stimulation (n = 50 cells). Boxes represent the 25th–75th percentiles and whiskers the 5th–95th percentiles