Figure 6. Hydrogen peroxide (H₂O₂) treatment decreases Rab5 membrane association on EE, increases early endosomal-mitochondrial contacts, and reduces transferrin uptake.

(A) Subcellular fractionation was performed in HeLa cells treated with H₂O₂ for 1 and 2 hr. The total membrane (M) fraction was obtained by centrifugation of the post nuclear supernatant at 200,000 g at 4°C for 1 hr, and supernatant was taken as cytosolic (C) fraction. Protein samples were loaded onto SDS-PAGE and imunoblotted with antibodies against Rab5, EEA1, GAPDH, and TOM20. The long exposure blot for EEA1 is shown (right panel). (B) Densitometric quantification of Rab5 in (A). Band intensities were calculated as normalized ratio between Rab5 to TOM20 in the M fraction, and Rab5 to GAPDH in the C fraction. Fold change is plotted on the y-axis. Error bars represent SEMs from three independent experiments. (C) BAC GFP-Rab5 HeLa cells were incubated with/out 250 µM H₂O₂ at 37°C for 2 hr. Cells were fixed and immunostained with EEA1 antibody. Colocalization analysis was performed between GFP-Rab5 and EEA1. (D) HeLa cells were seeded in a 384-well plate and pre-treated with either PBS (control) or 250 μM H₂O₂ at 37°C for 10 min. Cells were then pulsed with Alexa-647 Tfn (10 μg/ml) for 5 min, washed with PBS, fixed, and stained with DAPI (nuclear) and CellMask Blue (cytoplasmic) dyes. Images were acquired by the Yogokawa confocal microscope. Scale bars, 10 μm. (E) Quantification of the cytoplasmic fluorescence intensity per cell, n = 50. p Values based on two-tailed t-tests.