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. Author manuscript; available in PMC: 2009 Oct 1.

Published in final edited form as: Nat Cell Biol. 2009 Mar 8;11(4):468–476. doi: 10.1038/ncb1854

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under both normal and nutrient-starvation conditions in NIH 3T3 cells.

(b) Atg14L siRNA, as compared to control siRNA, decreased long-lived protein degradation in NIH 3T3 cells under both normal (p=0.007) and starvation (p=5E-6) conditions (asterisks, one-tailed Student’s t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA), a PI-3K inhibitor.

(c) Vps34 kinase assay. HEK 293T cells were co-transfected with myc-Vps34-Vps15 and FLAG-Atg14L or FLAG vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-myc antibody for the in vitro kinase assay. The resulting radioactive PtdIns(3)P was separated by thin layer chromatography (TLC) (lower left panel), quantified and normalized against the amount of immunoprecipitated myc-tagged Vps34 as measured by Western blot (upper left panel). The quantified results (right panel) showed that over-expressing Atg14L significantly up-regulated the Vps34 kinase activity by 2.5 folds, but only when Beclin 1 was also over-expressed (asterisk, p=0.04, one-tailed Student’s t-test with unequal variances, n=5).

(d) Co-localization of co-expressed Atg14L-EGFP (green) & Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar: 10 μm.

(e) Electron microscopic images show large structures (blue asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: (e1) concentric membrane “rings”; (e2) two large structures (3–5 μm in diameter) contain materials with high electron density; inset, enwrapping double membranes; (e3) numerous autophagosomes (blue arrows) in cytoplasm; (e4) immuno-electron microscopic image of a Atg14L-Beclin 1 structure (labeled with anti-GFP antibody and developed by DAB) enwrapped with concentric membrane “rings”. Abbreviations: M – mitochondria, N – nucleus. Scale bar: 500 nm.

(f-g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labeled by Atg14L-AsRed (red) and Beclin 1-myc (blue) in transfected HeLa cells. Some of these structures appeared to be “ring”-shaped (yellow arrows & inset). Scale bar: 10 μm.

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under both normal and nutrient-starvation conditions in NIH 3T3 cells.

(b) Atg14L siRNA, as compared to control siRNA, decreased long-lived protein degradation in NIH 3T3 cells under both normal (p=0.007) and starvation (p=5E-6) conditions (asterisks, one-tailed Student’s t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA), a PI-3K inhibitor.

(c) Vps34 kinase assay. HEK 293T cells were co-transfected with myc-Vps34-Vps15 and FLAG-Atg14L or FLAG vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-myc antibody for the in vitro kinase assay. The resulting radioactive PtdIns(3)P was separated by thin layer chromatography (TLC) (lower left panel), quantified and normalized against the amount of immunoprecipitated myc-tagged Vps34 as measured by Western blot (upper left panel). The quantified results (right panel) showed that over-expressing Atg14L significantly up-regulated the Vps34 kinase activity by 2.5 folds, but only when Beclin 1 was also over-expressed (asterisk, p=0.04, one-tailed Student’s t-test with unequal variances, n=5).

(d) Co-localization of co-expressed Atg14L-EGFP (green) & Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar: 10 μm.

(e) Electron microscopic images show large structures (blue asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: (e1) concentric membrane “rings”; (e2) two large structures (3–5 μm in diameter) contain materials with high electron density; inset, enwrapping double membranes; (e3) numerous autophagosomes (blue arrows) in cytoplasm; (e4) immuno-electron microscopic image of a Atg14L-Beclin 1 structure (labeled with anti-GFP antibody and developed by DAB) enwrapped with concentric membrane “rings”. Abbreviations: M – mitochondria, N – nucleus. Scale bar: 500 nm.

(f-g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labeled by Atg14L-AsRed (red) and Beclin 1-myc (blue) in transfected HeLa cells. Some of these structures appeared to be “ring”-shaped (yellow arrows & inset). Scale bar: 10 μm.