Context-dependent interactions between subunits of the mRNA decapping complex occur within P-bodies. (A) A549 cell lines stably co-expressing mNG-Dcp2 (green) and mScarlet-Dcp1A (magenta) were captured before and after treatment with 0.5 mM NaAsO2. (B) FLIM-phasor-guided masks were generated from mNG-Dcp2 and mScarlet-Dcp1A images to yield Mander’s colocalization coefficient before and after oxidative stress. Statistical significance was determined using a paired Student’s T test with mean and SE displayed. As a control, image tiles were randomized by shuffling prior colocalization analyses to ensure that Dcp2 and Dcp1A were not colocalizing by chance. (C) Mean FRET efficiencies between Dcp2 and Dcp1A in individual P-bodies are plotted with the mean highlighted (magenta). (A–C)N = 4 biological replicates with n = 194 cells. (D–G) Fluorescence intensity images of A549 cells expressing (D) mNG-EDC3 and mScarlet-Dcp1A or (n = 179 cells) (F) mNG-Dcp2 and mScarlet-EDC3 (n = 186 cells) were evaluated for condensate FLIM-FRET in panels E and G, respectively. N = 3 biological replicates. (C, E, and G) Statistical significance was determined by Student’s T test. ***P < 0.0001. All scale bars = 10 μm.