Figure 3.

Effect of MB in oxidative stress. (A, B) WT and MB KD MDA-MB-468 spheroids were challenged with different substances inducing oxidative stress. 24 h after transfection with Hs_MB_6 FlexiTube siRNA (KD) or control siRNA (WT), cells were either left untreated (control), irradiated with 5 Gy or treated with 500 µM nitric oxide (NO), 100 µM hydrogen peroxide (H₂O₂) or a combination of both for 4 h. Central necrosis of spheroids was visualized by DAPI intake. NO treatment led to an overall reduction in central necrotic cells whereas H₂O₂ treatment enhanced cellular death. Still, MB protected cells from dying in all tested conditions. Scale bar = 50 µm. Results are shown as pixels/field of view (FOV), mean +/- sd, n = 7-11 in 2 independent experiments, *p < 0.05 between genotypes; ^#p < 0.05 between treatment conditions. (C) 24 h after first transfection, WT and KD MDA-MB-468 monolayer cells were re-transfected with pC1-HyPer3, incubated for another 24 h under normoxia and finally challenged with 100 µM H₂O₂ to simulate hypoxic stress leading to an increased ROS formation. ROS levels were measured as changes in fluorescence of the HyPer3 plasmid during live cell imaging over a period of 270 s. Myoglobin knockdown led to significantly higher ROS levels in the cells after treatment with 100 µM H₂O₂. Mean +/- sd, n = 12 in 2 independent experiments.