Oxidative Bursts of Single Mitochondria Mediate Retrograde Signaling towards the ER

Fig1A: Whole field Airyscan fluorescence image of HepG2 cell loaded with TMRE imaged with 514nm excitation.
Fig1B&C: Images of cell from Fig1A + 6s/369s time lapse showing flicker events in single mitochondria, & uncropped difference images from inset. Difference images highlight changing TMRE signal red = decrease, blue = increase.
Fig1I: 490nm excitation fluorescence images of HepG2 cells loaded with Calcein-AM/Co2+ before/after 1200s treatment with ST +/- cyclosporine A (CsA).
Fig1K: Uncropped difference (TMRE) and overlay (Matrix-targeted SypHer) images. Overlay created from ratiometric imaging at 415nm & 485nm of SypHer. 415nm emission is colored green and 485nm red.

Fig2D: 2 kymograph images of TMRE (Upper: mpl-inferno LUT) & Grx1roGFP2 (Lower, Greenfireblue LUT). Source image of TMRE with segmented line ROI used to create kymograph images.
Fig2E: Uncropped difference (TMRE, upper) & overlay (Grx1roGFP2, lower) images of flicker event un unstimulated HepG2 cell. Overlay derived from 415nm (red; oxidized) & 485nm (green; reduced) emission from Grx1roGFP2. G: Uncropped difference & overlay images of flicker event from HepG2 cell stimulated with staurosporine (ST, 2µM).

Fig3E: Uncropped SypHer/RCaMP images covering the time period displayed. Difference images highlight changing SypHer ratio (Green = SypHer 485nm emission increase, red = decrease) & RCaMP emission (blue = increase, red = decrease). Time resolution is 2s/frame.
Fig3F: Original composite image created by integrating RCaMP 577nm emission signals and displaying result in 16_colors LUT overlaid onto grayscale cell image. Uncropped grayscale source images also included.

Fig4G: Original TMRE 577nm emission fluorescence images & FRET emission images presented in 16_colors LUT.

FigS1A: Uncropped 577nm emission images of TMRE stained HepG2 cells at t = 0 & t = 1200s in control (Ctrl) or staurosporine (ST) treated conditions.
FigS1B: Uncropped difference images of flicker event in TMRE stained HepG2 cell.

FigS4D: Uncropped composite images of time course presented. Images are of HepG2 cells stained with Hoechst 33321, CellEvent caspase substrate and TMRE imaged at 405,488 & 561nm excitation.
FigS4J: Uncropped FRET images presented in 16_colors LUT.