Figure S4. Quantification of diffusion... | Rockefeller University Press

$Quantification of diffusion and kinetic properties of HaloTagged autophagy proteins and autophagy induced foci. (A) Western blot showing partial depletion of CHMP2A in Halo-ATG13 cell line upon siRNA-mediated knockdown. (B) Graphs depicting foci frequency and foci lifetimes of ATG13 in complete media or EBSS treated with VPS34 inhibitor compound 31. Data represent mean ± SD. Letters indicate statistically homogenous groups established by ANOVA (P < 0.05). (C) Distribution of diffusion coefficients for Halo-ATG2A and Halo-ATG2A foci imaged a 3-s frame interval. In Halo-ATG13, Gaussian fitting shows two populations with distinct diffusive properties (solid purple and red lines). Cumulative fitting is shown in a solid black line. (D) Results of diffusive analysis for the parental Halo-ATG13 and ULK1, FIP200, and ATG101 knockout under control and EBSS starvation. Left panel depicts the percentage associated with each fraction. Right panels present the diffusion coefficients of the tracks based on the SpotON analysis. Boxes indicate confidence interval ± SD, the square indicates the average, and the horizontal line is the median; for each condition, three biological replicates were analyzed, ∼20 cells/replicate. (E) Images demonstrating the titration of the virus such that the G120A mutant does not form aggregates (left) and positive control in an ATG9A knockout (right). (F) Bar graph representing the average lifetime of GFP-P62 within ATG-edited cell lines. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). Letters indicate statistically homogenous groups established by ANOVA (P < 0.05). (G) Bar graph depicting the average lifetime of GFP-LC3 within ATG-edited cell lines. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). Letters indicate statistically homogenous groups established by ANOVA (P < 0.05). (H and I) Histograms of the lifetime of Halo-Tagged protein foci that colocalized (green) or did not colocalize (dark gray) with (H) GFP-P62 or (I) GFP-LC3 (light blue). Source data are available for this figure: SourceData FS4.$

Figure S4.

Quantification of diffusion and kinetic properties of HaloTagged autophagy proteins and autophagy induced foci. (A) Western blot showing partial depletion of CHMP2A in Halo-ATG13 cell line upon siRNA-mediated knockdown. (B) Graphs depicting foci frequency and foci lifetimes of ATG13 in complete media or EBSS treated with VPS34 inhibitor compound 31. Data represent mean ± SD. Letters indicate statistically homogenous groups established by ANOVA (P < 0.05). (C) Distribution of diffusion coefficients for Halo-ATG2A and Halo-ATG2A foci imaged a 3-s frame interval. In Halo-ATG13, Gaussian fitting shows two populations with distinct diffusive properties (solid purple and red lines). Cumulative fitting is shown in a solid black line. (D) Results of diffusive analysis for the parental Halo-ATG13 and ULK1, FIP200, and ATG101 knockout under control and EBSS starvation. Left panel depicts the percentage associated with each fraction. Right panels present the diffusion coefficients of the tracks based on the SpotON analysis. Boxes indicate confidence interval ± SD, the square indicates the average, and the horizontal line is the median; for each condition, three biological replicates were analyzed, ∼20 cells/replicate. (E) Images demonstrating the titration of the virus such that the G120A mutant does not form aggregates (left) and positive control in an ATG9A knockout (right). (F) Bar graph representing the average lifetime of GFP-P62 within ATG-edited cell lines. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). Letters indicate statistically homogenous groups established by ANOVA (P < 0.05). (G) Bar graph depicting the average lifetime of GFP-LC3 within ATG-edited cell lines. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). Letters indicate statistically homogenous groups established by ANOVA (P < 0.05). (H and I) Histograms of the lifetime of Halo-Tagged protein foci that colocalized (green) or did not colocalize (dark gray) with (H) GFP-P62 or (I) GFP-LC3 (light blue). Source data are available for this figure: SourceData FS4.

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