Figure 4.
Figure 4. ATG9A modulates the distribution of the cytoplasmic PI4P pool and PI4KIIIβ. (A) Scatterplot of proteins associated with ATG9A-positive membranes from HEK293A CTRL or CrARFIP2 KO (clone 1) cells incubated in ES. The x axis displays the Log2 of the SILAC ratio (CrARFIP2 KO/CTRL), and the y axis displays the Log10 of the intensity. Highlighted in black and green are ATG9A and ARFIP2, respectively. Highlighted in red are proteins from the PI metabolic process GO category in ATG9A-positive membranes; yellow are proteins related to the production of PI4P depleted from ATG9A-positive membrane in CrARFIP2 KO (clone 1) cells. See Table S3. (B) CTRL or CrARFIP2 KO (clone 1) cells were incubated in ES for 2 h, and the ATG9A-positive compartment was immunoisolated before immunoblot for ATG9A, PI4KΙΙα, and PI4KIIIβ. Left: *, Nonspecific band. Quantification of the relative amount of PI4KIIIβ after normalization to immunoisolated ATG9A. Statistical analysis using two-tailed unpaired Student’s t test, mean ± SEM, n = 3 experiments; *, P ≤ 0.05; ***, P ≤ 0.001. (C) HEK293A cells were treated with RF siRNA or ATG9A siRNA for 72 h and then incubated in FM or ES for 2 h before immunostaining for PI4P and GM130. Scale bars, 10 µm. (D) Quantification of PI4P and GM130 colocalization in C. Mean ± SEM, n = 3 experiments, Pearson’s coefficient of 30 cells per condition per independent experiment was quantified, statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test; ***, P ≤ 0.001. (E) HEK293A cells were treated with RF or ATG9A siRNA for 72 h and then incubated in FM or ES for 2 h before immunostaining for PI4KIIIβ and GM130. Scale bars, 10 µm. (F) Quantification of PI4KIIIβ and GM130 colocalization in E. Mean ± SEM, n = 3 experiments, Pearson’s coefficient of 30 cells per condition per independent experiment was quantified, statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test; *, P ≤ 0.05; ***, P ≤ 0.001.

ATG9A modulates the distribution of the cytoplasmic PI4P pool and PI4KIIIβ. (A) Scatterplot of proteins associated with ATG9A-positive membranes from HEK293A CTRL or CrARFIP2 KO (clone 1) cells incubated in ES. The x axis displays the Log2 of the SILAC ratio (CrARFIP2 KO/CTRL), and the y axis displays the Log10 of the intensity. Highlighted in black and green are ATG9A and ARFIP2, respectively. Highlighted in red are proteins from the PI metabolic process GO category in ATG9A-positive membranes; yellow are proteins related to the production of PI4P depleted from ATG9A-positive membrane in CrARFIP2 KO (clone 1) cells. See Table S3. (B) CTRL or CrARFIP2 KO (clone 1) cells were incubated in ES for 2 h, and the ATG9A-positive compartment was immunoisolated before immunoblot for ATG9A, PI4KΙΙα, and PI4KIIIβ. Left: *, Nonspecific band. Quantification of the relative amount of PI4KIIIβ after normalization to immunoisolated ATG9A. Statistical analysis using two-tailed unpaired Student’s t test, mean ± SEM, n = 3 experiments; *, P ≤ 0.05; ***, P ≤ 0.001. (C) HEK293A cells were treated with RF siRNA or ATG9A siRNA for 72 h and then incubated in FM or ES for 2 h before immunostaining for PI4P and GM130. Scale bars, 10 µm. (D) Quantification of PI4P and GM130 colocalization in C. Mean ± SEM, n = 3 experiments, Pearson’s coefficient of 30 cells per condition per independent experiment was quantified, statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test; ***, P ≤ 0.001. (E) HEK293A cells were treated with RF or ATG9A siRNA for 72 h and then incubated in FM or ES for 2 h before immunostaining for PI4KIIIβ and GM130. Scale bars, 10 µm. (F) Quantification of PI4KIIIβ and GM130 colocalization in E. Mean ± SEM, n = 3 experiments, Pearson’s coefficient of 30 cells per condition per independent experiment was quantified, statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test; *, P ≤ 0.05; ***, P ≤ 0.001.

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