Figure 6. Low concentrations of metformin reduce reactive oxygen species in senescence via suppression of NOX4.
(A) Human fibroblasts were irradiated with 20 Gy and treated with 100 µM metformin for 10 days. Left: Representative images of cells treated as indicated, red: NADPH oxidase 4 (NOX4) immunofluorescence, blue: DAPI. Right: Quantification of NOX4 fluorescence intensity. (B) Left: Representative NOX4 Western blot. Cells treated as above. A complete blot is provided as Figure 6—source data 1. Right: Average NOX4 signal intensity, normalised to total protein. (C) Left: Sen-β-Gal staining in EGFP- and NOX4-overexpressing fibroblasts. Right: Average frequencies of Sen-β-Gal-positive cells. (D) Left: Dihydroethidium (DHE) staining in EGFP- and NOX4-overexpressing fibroblasts. DHE fluorescence in red, cells visualised in phase contrast. Right: Average DHE fluorescence intensity per cell. (E) Co-staining for NOX4 (red) and IL-6 (green) on EGFP- or NOX4-transfected fibroblasts. Right: Fluorescence intensity levels for EGFP (top), NOX4 (middle), and IL-6 in EGFP- and NOX4-transfected cells. (F) Correlation between cellular NOX4 and IL6 fluorescence signals in NOX4-transfected cells. (G) Correlation between cellular EGFP and IL6 fluorescence signals in EGFP-transfected cells. (H) Left: Sen-β-Gal staining in senescent fibroblasts treated with the indicated concentrations of diphenyleneiodonium chloride (DPI). Right: Frequencies of Sen-β-Gal-positive cells. (I) Left: DHE staining in senescent fibroblasts treated with the indicated concentrations of DPI. DHE fluorescence in red, cells visualised in phase contrast. Right: Average intensity of DHI fluorescence per cell. All experiments N ≥3.
Figure 6—figure supplement 1. NADPH oxidase 4 (NOX4) does not colocalise with mitochondria.
