All tracked populations of autophagy factors foci require ULK1 and PI3K activity. (A) Fluorescence gel and Western blots demonstrating successful knockout of ULK1, FIP200, and ATG101 from the Halo-ATG13 cell line. (B) Western blots demonstrating impaired autophagy when ULK1, FIP200, and ATG101 are individually depleted from the Halo-ATG13 cell line. For the treatment experiments, cells were preincubated in control media with ULK1-101 (1 μM) or without drug for 1 h where indicated. Cells were then switched to their control or EBSS starvation media, with or without bafilomycin (100 nM), for an additional hour. (C) Quantification of the Western blots in B. Data represent mean ± SD over three biological replicates. Phospho-P62 band (red striped bar graph) was detected and quantified only in the FIP200 and ATG101 knock-out cell lines. (D) Histograms of Halo-ATG13 foci lifetimes for the parental Halo-ATG13 cells and ULK1, FIP200, ATG101 knock-out cell lines in control conditions (Control) and after 1 h nutrient starvation (EBSS). Three biological replicates (20–30 cells per replicate) were performed for each cell line. The number of data points (n) is indicated in each graph in the figure panels. (E) Quantification of the number of foci formed per cell by HaloTag-ATG13 over the course of 1 h imaging in control (Control) and nutrient starvation (EBSS) conditions (N = 3, mean ± SD). A two-tailed t test was used for statistical analysis. (F) Histograms of foci lifetimes for the HaloTag-ATG2A (left) and HaloTag-ATG13 (center) in control and nutrient starvation (EBSS, 1 h) conditions with and without wortmannin (1 μM). Cells were pretreated with Wortmannin for 1 h. Right panel presents the quantification of the foci frequency. Data represent mean ± SD over three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis (*P < 0.05, ***P < 0.001).
Figure 5.

All tracked populations of autophagy factors foci require ULK1 and PI3K activity. (A) Fluorescence gel and Western blots demonstrating successful knockout of ULK1, FIP200, and ATG101 from the Halo-ATG13 cell line. (B) Western blots demonstrating impaired autophagy when ULK1, FIP200, and ATG101 are individually depleted from the Halo-ATG13 cell line. For the treatment experiments, cells were preincubated in control media with ULK1-101 (1 μM) or without drug for 1 h where indicated. Cells were then switched to their control or EBSS starvation media, with or without bafilomycin (100 nM), for an additional hour. (C) Quantification of the Western blots in B. Data represent mean ± SD over three biological replicates. Phospho-P62 band (red striped bar graph) was detected and quantified only in the FIP200 and ATG101 knock-out cell lines. (D) Histograms of Halo-ATG13 foci lifetimes for the parental Halo-ATG13 cells and ULK1, FIP200, ATG101 knock-out cell lines in control conditions (Control) and after 1 h nutrient starvation (EBSS). Three biological replicates (20–30 cells per replicate) were performed for each cell line. The number of data points (n) is indicated in each graph in the figure panels. (E) Quantification of the number of foci formed per cell by HaloTag-ATG13 over the course of 1 h imaging in control (Control) and nutrient starvation (EBSS) conditions (N = 3, mean ± SD). A two-tailed t test was used for statistical analysis. (F) Histograms of foci lifetimes for the HaloTag-ATG2A (left) and HaloTag-ATG13 (center) in control and nutrient starvation (EBSS, 1 h) conditions with and without wortmannin (1 μM). Cells were pretreated with Wortmannin for 1 h. Right panel presents the quantification of the foci frequency. Data represent mean ± SD over three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis (*P < 0.05, ***P < 0.001).

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