Figure S5.
CRISPR/Cas9 KO IRAK4 and IRAK1 EL4 cell lines, and MyD88-GFP dynamics in KO cell lines. (A and B) Validation of IRAK4 and IRAK1 KO cell lines. Western blot analysis of two monoclonal EL4 IRAK4 KO (A) and IRAK1 KO (B) cell lines. (C) The percentage per cell of large MyD88 oligomers in WT, IRAK4 KO, and IRAK1 KO EL4 cell lines. Quantification of the percentage (%) per cell of MyD88-GFP puncta with a maximum intensity ≥4.5× GFP. Violin plots show the distribution of the cell data. Data points superimposed on violin plots are the averages of independent experimental replicates. Bars represent mean ± SEM (n = 3 or 4 experimental replicates, with >10 cells measured per replicate). (D) The percentage per cell of large MyD88 oligomers is equivalent for short- and long-lived MyD88-GFP puncta across WT and KO cell lines. Quantification of the proportion (%) per cell of MyD88-GFP puncta with a maximum intensity of ≥4.5× GFP categorized by lifetimes of <50 s or ≥50 s. Violin plots show the distribution of the cell data. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 3 or 4 experimental replicates, with >10 cells measured per replicates). WT and KO image means were compared using an unpaired Student's t test. (E) IL2 release in EL4-WT cells treated with IRAK4 inhibitor. EL4 cells were pretreated with DMSO or IRAK4 inhibitor (20 µm) for 30 min. Cells were then left untreated or stimulated with 10 ng/ml of IL1 for a further 24 h. IL2 release was assayed by ELISA. Values for IRAK4 inhibitor cells shown relative to DMSO-only treated cells. Average values calculated from three independent experiments. Bars represent mean ± SEM. (F) 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 experimental replicates; encompasses measurements from 19–39 cells). Bars represent mean ± SEM. P values were calculated using a two-tailed unpaired Student’s t test. (G) Distribution of lifetimes for tracked MyD88-GFP puncta with a maximum intensity of <4.5× or ≥4.5× GFP in DMSO control (n = 91,876 for <4.5× GFP and n = 4,836 for ≥4.5× GFP), and IRAK4 inhibitor (n = 64,822 for <4.5× GFP and n = 3,828 for ≥4.5× GFP) –treated cells. MyD88-GFP puncta lifetimes collated from three experimental replicates. Max, maximum; Norm., normalized; MW, molecular weight.

CRISPR/Cas9 KO IRAK4 and IRAK1 EL4 cell lines, and MyD88-GFP dynamics in KO cell lines. (A and B) Validation of IRAK4 and IRAK1 KO cell lines. Western blot analysis of two monoclonal EL4 IRAK4 KO (A) and IRAK1 KO (B) cell lines. (C) The percentage per cell of large MyD88 oligomers in WT, IRAK4 KO, and IRAK1 KO EL4 cell lines. Quantification of the percentage (%) per cell of MyD88-GFP puncta with a maximum intensity ≥4.5× GFP. Violin plots show the distribution of the cell data. Data points superimposed on violin plots are the averages of independent experimental replicates. Bars represent mean ± SEM (n = 3 or 4 experimental replicates, with >10 cells measured per replicate). (D) The percentage per cell of large MyD88 oligomers is equivalent for short- and long-lived MyD88-GFP puncta across WT and KO cell lines. Quantification of the proportion (%) per cell of MyD88-GFP puncta with a maximum intensity of ≥4.5× GFP categorized by lifetimes of <50 s or ≥50 s. Violin plots show the distribution of the cell data. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 3 or 4 experimental replicates, with >10 cells measured per replicates). WT and KO image means were compared using an unpaired Student's t test. (E) IL2 release in EL4-WT cells treated with IRAK4 inhibitor. EL4 cells were pretreated with DMSO or IRAK4 inhibitor (20 µm) for 30 min. Cells were then left untreated or stimulated with 10 ng/ml of IL1 for a further 24 h. IL2 release was assayed by ELISA. Values for IRAK4 inhibitor cells shown relative to DMSO-only treated cells. Average values calculated from three independent experiments. Bars represent mean ± SEM. (F) 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 experimental replicates; encompasses measurements from 19–39 cells). Bars represent mean ± SEM. P values were calculated using a two-tailed unpaired Student’s t test. (G) Distribution of lifetimes for tracked MyD88-GFP puncta with a maximum intensity of <4.5× or ≥4.5× GFP in DMSO control (n = 91,876 for <4.5× GFP and n = 4,836 for ≥4.5× GFP), and IRAK4 inhibitor (n = 64,822 for <4.5× GFP and n = 3,828 for ≥4.5× GFP) –treated cells. MyD88-GFP puncta lifetimes collated from three experimental replicates. Max, maximum; Norm., normalized; MW, molecular weight.

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