Fig. 4

Effects of intracellular miR-21 knockdown in ameliorating ROS homeostasis in increasing the antioxidant response in HUVECs. a KRIT1 mRNA expression levels were reduced in OG and HG, whereas anti-miR-21 reversed this effect. b SOD2 mRNA was unchanged during glucose concentration changes, but the level was increased during miR-21 silencing. c Endogenous KRIT1 staining was reduced in OG and HG. Silencing miR-21 increased KRIT1 fluorescence intensity. Fluorescence images are representative of n = 6 per experiments. Original magnification ×20; scale bar, 20 μm. d Densitometry of KRIT1 images measured by the ratio of the % area of positive staining and nuclei count (DAPI) per field (at least n = 6 per condition). e–h Whole cell lysates densitometry of (E) SOD2 protein expression levels, phosphorylation of f serine-40 on NRF2 and g ERK1/2. Normalisation was to actin-beta (ACT-b) and to total amounts of ERK1/2 and NRF2, respectively for phosphorylated status. h Representative immunoblots of HUVECs under NG, OG and HG, before and after transfection with an anti-miR-21 inhibitor. i–k miR-21 expression levels during administration of ROS scavenger, alpha lipoic acid (aLA), that reduced miR-21 expression and j conversely increased SOD2 protein levels; k representative immunoblots of SOD2 normalised to ACT-b levels. Data are mean (± SEM), *p < 0.05, **p < 0.01, and ***p < 0.001 vs control; ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 and ^††††p < 0.0001 vs OG, ^§p < 0.05, ^§§p < 0.01, ^§§§p < 0.001 and ^§§§§p < 0.0001 vs HG