Extended Data Fig. 2: Validating targeting specificity and cellular uptake with fluorescent control compounds.
The targeting specificity of fluorogenic RTAs cannot directly validated by colocalization studies, as they can only be visualized after reacting with lipid peroxyl radicals. a-e. 100x HILO images of HT-1080 cells co-stained with H4B-CH3 (10 nM) and either ERTracker Red (1 μM, a) or LipidSpot 610 (1x, b) to validate ER and lipid droplets accumulation, respectively, of H4B-PMHC. c) mito-H5B-CH3 (10 nM) and mitoTracker DR (25 nM) to show mitochondrial localization of mito-H5B-PMHC. d) lyso-H4B-CH3 (10 nM) and lysoTracker DR (75 nM) to show lysosomal localization of lyso-H4B-PMHC. e) PM-H4B-CH3 (100 nM) in HT-1080 cells transfected with PM-RFP to show PM localization of PM-H4B-PMHC. The region within the yellow outline was used to calculate Mander’s and Pearson’s coefficients. In each experiment, cells stained with the commercial organelle marker were incubated with the fluorescent control compound for 10 minutes, before colocalization images were captured. Each experiment was conducted once. f) The intensity of the 488-channel along the indicated line is plotted against that of the 561 channel to show colocalization of PM-H4B-CH3 with PM-RFP. g) The cell outline (blue) was identified via DIC channel and was plotted against the intensity of the 488-channel along the indicated line. Scale bar = 12 µm. h) Mander’s coefficients (M1 and M2) and Pearson’s coefficient of the fluorescent control compounds with commercial organelle markers. i) Time course of partition profile of the fluorescent control compounds in HT-1080 cells. Imaging was initiated immediately after addition of the fluorescent control compound. Bars = SEM, n = 4 (replicates across independent wells).
