Figure 4. Enrichment of ATG9A in the... | Rockefeller University Press

Figure 4.

$Enrichment of ATG9A in the autophagosome fraction is dependent on autophagosome biogenesis machinery. HEK293 cells were gene-edited to knockout autophagy genes involved in autophagosome biogenesis and then fractionated as in Fig. 3. (A–D) Immunoblot of cell membrane fractions from indicated knockout cells. Cells in A–D were grown in DMEM under basal conditions. Loaded protein: 3 µg. (E) Densitometric quantification of ATG9A, LC3B-II, Calnexin, and TOM20 in each cell line membrane fractionation displayed as average ± SD. The intensity of the bands in A–D were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. All KO cell lines, n = 3 biological replicates. TOM20 signal was oversaturated in fraction 5 of the FIP200 KO in one replicate, so n = 2 included biological replicates for TOM20 in FIP200 KO fraction 5. ATG9A is enriched in fraction 5 when initiation or lipidation is inhibited but not enriched over lysate in the AP fraction. Very little AP fraction was recovered for any sample (Table S1) and total LC3B-II in lysate is minimal (A and B) or lost (C and D). Where trace LC3B-II is present, there is a clear enrichment in the AP fraction. (F) Densitometric quantification of ATG9A enrichment in fraction 6 compared across all cell lines tested displayed as average ± SD. These values were taken directly from E, Fig. 3, B and C; and Fig. S4 B. WT Starved+Baf, n = 4 biological replicates; all other cell lines, n = 3 biological replicates. (G) Densitometric quantification of ATG9A total recovery in fraction 6 compared across all cell lines tested with the WT Starved+Baf condition as the reference value (100% recovery) displayed as average ± SD. These values were generated by multiplying the values in F by the fraction recovery highlighted in Table S1. WT Starved+Baf, n = 4 biological replicates; all other cell lines, n = 3 biological replicates.$

Enrichment of ATG9A in the autophagosome fraction is dependent on autophagosome biogenesis machinery. HEK293 cells were gene-edited to knockout autophagy genes involved in autophagosome biogenesis and then fractionated as in Fig. 3. (A–D) Immunoblot of cell membrane fractions from indicated knockout cells. Cells in A–D were grown in DMEM under basal conditions. Loaded protein: 3 µg. (E) Densitometric quantification of ATG9A, LC3B-II, Calnexin, and TOM20 in each cell line membrane fractionation displayed as average ± SD. The intensity of the bands in A–D were normalized to the lysate, and statistical significance was assessed by two-way ANOVA. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. All KO cell lines, n = 3 biological replicates. TOM20 signal was oversaturated in fraction 5 of the FIP200 KO in one replicate, so n = 2 included biological replicates for TOM20 in FIP200 KO fraction 5. ATG9A is enriched in fraction 5 when initiation or lipidation is inhibited but not enriched over lysate in the AP fraction. Very little AP fraction was recovered for any sample (Table S1) and total LC3B-II in lysate is minimal (A and B) or lost (C and D). Where trace LC3B-II is present, there is a clear enrichment in the AP fraction. (F) Densitometric quantification of ATG9A enrichment in fraction 6 compared across all cell lines tested displayed as average ± SD. These values were taken directly from E, Fig. 3, B and C; and Fig. S4 B. WT Starved+Baf, n = 4 biological replicates; all other cell lines, n = 3 biological replicates. (G) Densitometric quantification of ATG9A total recovery in fraction 6 compared across all cell lines tested with the WT Starved+Baf condition as the reference value (100% recovery) displayed as average ± SD. These values were generated by multiplying the values in F by the fraction recovery highlighted in Table S1. WT Starved+Baf, n = 4 biological replicates; all other cell lines, n = 3 biological replicates.