Fig. 2

Mild-oxidative stress (H₂O₂ 25 µM) induced the targeting of the eMI-reporter Venus to late endosomes through an eMI-like process in RT cells. (A) Representative images of RTH-149 cells stably expressing an eMI-reporter consisting in a KFERQ recognition motif fused to either the N-terminal or C-terminal of split Venus and visualized by fluorescence microscopy. The exposure to H₂O₂ 25 µM, for either 8 h or 16 h, induced the reconstitution of the Venus fluorescent protein (green) and the formation of Venus-puncta, in comparison with the CT, where the reporter showed a diffuse distribution with only discrete and scattered puncta standing out from the background noise. Nuclei were stained with DAPI (blue) and the inset show an amplified region of each image. (B) Quantification of KFERQ-Venus number of puncta per cell. All values correspond to individual images (CT 63; 8 h H₂O₂ 63; 16 h H₂O₂ 63), with > 18 images/experiment in a total of 3 independent experiments (> 600 cells per condition). Different letters denote significant differences between groups compared by the non-parametric Kruskal–Wallis test (p < 0.0001) followed by Dunn’s multiple comparisons tests. All data are presented as Mean ± SEM; scale bars: 10 µm. (C) Representative images of H₂O₂-induced KFERQ-Venus puncta (green) in cells transiently transfected with RAB7-RFP (red) visualized by fluorescence microscopy. Most of the KFERQ-Venus puncta co-localized with the red late endosome marker RAB7-RFP, as indicated by the yellow arrowheads and amplified in the insets, and as supported by the positive Pearson’s correlation coefficient (PCC) results of the global analysis of 85 single cells using the BIOP version of JACoP plugin for Fiji. Scale bars: 10 µm. (D) Representative immunoelectron microscopy images of H₂O₂-incubated RTH-149 cells expressing the eMI sensor (Venus) using anti-Venus antibody, showing Venus immunoreactivity (arrowheads in the amplified insets) within MVBs. Scale bars: 2 µm or 0.5 µm (insets). (E) Other two representative IEM images at a higher magnification showing Venus immunoreactivity (arrowheads) within MVBs. Scale bar: 200 nm.