IRAK4 KO leads to super MyD88 oligomers. (A–C) TIRF images of MyD88-GFP in EL4 WT (A), IRAK1 KO (B), and IRAK4 KO (C) cells. Time-series TIRF images from the region of interest (white box) showing representative MyD88 puncta. A fluorescence intensity time trace from each time series is shown below. (D) Quantification of the maximum intensity of MyD88-GFP puncta per cell with lifetimes of <50 s or ≥50 s. Violin plots show the distribution of individual cell measurements. Colored dots superimposed on violin plots correspond to the average value in the independent experiments (n = 3 or 4 experimental replicates; encompasses measurements from 10 to 47 cells). Bars represent mean ± SEM. P values were calculated using a two-tailed unpaired Student's t test. (E) Distribution of lifetimes for tracked MyD88-GFP puncta with a maximum intensity of <4.5× or ≥4.5× GFP in WT (n = 73,180 for <4.5× GFP and n = 24,631 for ≥4.5× GFP), IRAK4 KO (n = 18,478 for <4.5× GFP and n = 9,795 for ≥4.5× GFP), and IRAK1 KO (n = 63,382 for <4.5× GFP and n = 19,735 for ≥4.5× GFP) and IRAK4 KO + IRAK4-mScarlet (n = 18,764 for <4.5× GFP and n = 5,627 for ≥4.5× GFP) cells. MyD88-GFP puncta lifetimes collated from 3 or 4 experimental replicates. (F) 2D histogram of MyD88-GFP puncta lifetime versus change in fluorescent intensity for WT (n = 64,149), IRAK4 KO (n = 13,960), IRAK1 KO (n = 54,551), and IRAK4 KO + IRAK4-mScarlet (n = 18,154) cells. Linear fit is shown as a blue line. The coefficient used is Spearman’s rank correlation coefficient. Max, maximum; Norm., normalized.