Figure S3.
Analysis of MyD88-GFP puncta size, lifetime, and correlation analysis from biological replicates of Fig. 3. (A) Size distribution of MyD88-GFP puncta from additional experimental replicates. Density plot of the maximum fluorescence intensity of MyD88-GFP puncta (dark blue; replicate 1, n = 1,952 puncta from 16 cells; replicate 2, n = 7,637 puncta from 19 cells; replicate 3, n = 11,973 puncta from 24 cells). For comparison, we included the intensity distribution of single GFP fluorophores (green; replicate 1, n = 298,293 GFP particles; replicate 2, n = 7,995 GFP particles; replicate 3, n = 7,995 GFP particles). To estimate the distribution of a 6× GFP multimer (light blue), a Gaussian curve was fitted to the 1× GFP intensity distribution (see Materials and methods). Blue background shade indicates ≥4.5× GFP. (B) Proportion (%) of the maximum intensity (<4.5× or ≥4.5× GFP) of MyD88-GFP puncta by cell across experimental replicates. Data points are the proportion of the maximum intensity <4.5× GFP (red) or ≥4.5× GFP (blue) by individual cells from six independent experiments. Percentage is the replicate’s proportion of MyD88-GFP puncta with maximum intensity ≥4.5× GFP, n = cell, replicates 1–6: 16% n = 6; 16%, n = 14; 3%, n = 13; 17%, n = 16; 13%, n = 19; 17%, n = 24. (C) Lifetime distribution of MyD88-GFP puncta from additional experimental replicates. MyD88-GFP lifetime histogram for MyD88-GFP puncta with maximum intensity <4.5× GFP (red) or ≥4.5× GFP (blue; replicate 1, n = 1,616 puncta <4.5× GFP and n = 336 ≥4.5× GFP; replicate 2, n = 6,553 puncta <4.5× GFP and n = 1,084 puncta ≥4.5× GFP; replicate 3, n = 9,913 puncta <4.5× GFP, and n = 2,060 puncta ≥4.5× GFP). Puncta count is in log scale. (D) Proportion (%) of the lifetimes (<50 s or ≥50 s) that are bright MyD88-GFP puncta (≥4.5× GFP) in individual cells across experimental replicates. Data points are the proportion of individual cells from independent experimental replicates. Percentage of MyD88-GFP puncta with a maximum intensity of ≥4.5× MyD88 that are <50 s (n = cells, from replicate 1–6): 7%, n = 14; 13%, n = 6; 2%, n = 13; 8%, n = 16; 7%, n = 19; 9%, n = 24). Percentage of MyD88-GFP puncta with a maximum intensity of ≥4.5×x GFP that have lifetimes ≥50 s (n = cells, replicate 1–6): 59%, n = 14; 67%, n = 6; 88%, n = 13; 83%, n = 16; 52%, n = 19; 67%, n = 24). Long-lived events are more likely to be brighter. Bars in B and D represent the replicate mean. (E) Correlation between lifetime and intensity growth of MyD88-GFP puncta from additional experimental replicates. 2D histogram of MyD88-GFP puncta lifetime by change in fluorescence intensity (calculated as maximum intensity minus starting intensity). MyD88-GFP puncta with longer lifetimes have a greater increase in fluorescence intensity. Linear regression line is shown in blue with a 95% CI in gray. There is a statistically significant strong positive correlation between lifetime and growth (n = puncta, replicate 1–3: R = 0.58, P < 0.001, n = 1,952; R = 0.62, P < 0.001, n = 7,637; R = 0.62, P < 0.001, n = 11,973). Correlations are Spearman’s rank correlation coefficient. Puncta count is shown in log scale. (F) TIRF images of IL1β-JF646 and MyD88-GFP showing MyD88 puncta formation at a membrane density 0.1 IL1β per square micrometer. Fluo, fluorescence; Norm., normalized.

Analysis of MyD88-GFP puncta size, lifetime, and correlation analysis from biological replicates of Fig. 3. (A) Size distribution of MyD88-GFP puncta from additional experimental replicates. Density plot of the maximum fluorescence intensity of MyD88-GFP puncta (dark blue; replicate 1, n = 1,952 puncta from 16 cells; replicate 2, n = 7,637 puncta from 19 cells; replicate 3, n = 11,973 puncta from 24 cells). For comparison, we included the intensity distribution of single GFP fluorophores (green; replicate 1, n = 298,293 GFP particles; replicate 2, n = 7,995 GFP particles; replicate 3, n = 7,995 GFP particles). To estimate the distribution of a 6× GFP multimer (light blue), a Gaussian curve was fitted to the 1× GFP intensity distribution (see Materials and methods). Blue background shade indicates ≥4.5× GFP. (B) Proportion (%) of the maximum intensity (<4.5× or ≥4.5× GFP) of MyD88-GFP puncta by cell across experimental replicates. Data points are the proportion of the maximum intensity <4.5× GFP (red) or ≥4.5× GFP (blue) by individual cells from six independent experiments. Percentage is the replicate’s proportion of MyD88-GFP puncta with maximum intensity ≥4.5× GFP, n = cell, replicates 1–6: 16% n = 6; 16%, n = 14; 3%, n = 13; 17%, n = 16; 13%, n = 19; 17%, n = 24. (C) Lifetime distribution of MyD88-GFP puncta from additional experimental replicates. MyD88-GFP lifetime histogram for MyD88-GFP puncta with maximum intensity <4.5× GFP (red) or ≥4.5× GFP (blue; replicate 1, n = 1,616 puncta <4.5× GFP and n = 336 ≥4.5× GFP; replicate 2, n = 6,553 puncta <4.5× GFP and n = 1,084 puncta ≥4.5× GFP; replicate 3, n = 9,913 puncta <4.5× GFP, and n = 2,060 puncta ≥4.5× GFP). Puncta count is in log scale. (D) Proportion (%) of the lifetimes (<50 s or ≥50 s) that are bright MyD88-GFP puncta (≥4.5× GFP) in individual cells across experimental replicates. Data points are the proportion of individual cells from independent experimental replicates. Percentage of MyD88-GFP puncta with a maximum intensity of ≥4.5× MyD88 that are <50 s (n = cells, from replicate 1–6): 7%, n = 14; 13%, n = 6; 2%, n = 13; 8%, n = 16; 7%, n = 19; 9%, n = 24). Percentage of MyD88-GFP puncta with a maximum intensity of ≥4.5×x GFP that have lifetimes ≥50 s (n = cells, replicate 1–6): 59%, n = 14; 67%, n = 6; 88%, n = 13; 83%, n = 16; 52%, n = 19; 67%, n = 24). Long-lived events are more likely to be brighter. Bars in B and D represent the replicate mean. (E) Correlation between lifetime and intensity growth of MyD88-GFP puncta from additional experimental replicates. 2D histogram of MyD88-GFP puncta lifetime by change in fluorescence intensity (calculated as maximum intensity minus starting intensity). MyD88-GFP puncta with longer lifetimes have a greater increase in fluorescence intensity. Linear regression line is shown in blue with a 95% CI in gray. There is a statistically significant strong positive correlation between lifetime and growth (n = puncta, replicate 1–3: R = 0.58, P < 0.001, n = 1,952; R = 0.62, P < 0.001, n = 7,637; R = 0.62, P < 0.001, n = 11,973). Correlations are Spearman’s rank correlation coefficient. Puncta count is shown in log scale. (F) TIRF images of IL1β-JF646 and MyD88-GFP showing MyD88 puncta formation at a membrane density 0.1 IL1β per square micrometer. Fluo, fluorescence; Norm., normalized.

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