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Figure S3. Colocalization of GFP-DFCP1 with autophagy-related proteins and the ER. (A) HEK-293 clones expressing GFP-DFCP1 were left untreated or starved for 60 min and then stained for endogenous MAP-LC3. Note that both GFP-DFCP1 and MAP-LC3 translocated to punctate structures during starvation. 50% of endogenous MAP-LC3 puncta colocalized with GFP-DFCP1. A series of colocalization examples during starvation, which are analogous to the interactions seen during live imaging, are shown in panels a–h of the merged image and as separate magnified panels. (B) HEK-293 cells or clones expressing GFP-DFCP1 were left untreated or starved for 60 min and then stained for endogenous Atg5. Note that in both cell types, Atg5 became more punctate during starvation, and these punctate structures colocalized with GFP-DFCP1. These images were obtained using wide-field microscopy. Arrows highlight examples of Atg5 punctae interacting with (or at least in very close proximity to) DFCP1 punctae/rings formed following starvation. (C) Additional examples of the colocalization of GFP-DFCP1 with Atg5 after amino acid starvation using confocal microscopy. Both sets of photographs are from cells starved for 60 min; 80% of DFCP1-positive puncta colocalized with endogenous Atg5 puncta. A series of colocalization examples are shown magnified in panels 1–3. (D) HEK-293 clones expressing GFP-DFCP1 were transfected with dsRed-ER and imaged live by confocal microscopy during amino acid starvation. Shown in panels A–E are examples from several such videos, where the DFCP1 omegasomes coincide with ER regions (boxed areas).
Figure S3. Colocalization of GFP-DFCP1 with autophagy-related proteins and the ER. (A) HEK-293 clones expressing GFP-DFCP1 were left untreated or starved for 60 min and then stained for endogenous MAP-LC3. Note that both GFP-DFCP1 and MAP-LC3 translocated to punctate structures during starvation. 50% of endogenous MAP-LC3 puncta colocalized with GFP-DFCP1. A series of colocalization examples during starvation, which are analogous to the interactions seen during live imaging, are shown in panels a–h of the merged image and as separate magnified panels. (B) HEK-293 cells or clones expressing GFP-DFCP1 were left untreated or starved for 60 min and then stained for endogenous Atg5. Note that in both cell types, Atg5 became more punctate during starvation, and these punctate structures colocalized with GFP-DFCP1. These images were obtained using wide-field microscopy. Arrows highlight examples of Atg5 punctae interacting with (or at least in very close proximity to) DFCP1 punctae/rings formed following starvation. (C) Additional examples of the colocalization of GFP-DFCP1 with Atg5 after amino acid starvation using confocal microscopy. Both sets of photographs are from cells starved for 60 min; 80% of DFCP1-positive puncta colocalized with endogenous Atg5 puncta. A series of colocalization examples are shown magnified in panels 1–3. (D) HEK-293 clones expressing GFP-DFCP1 were transfected with dsRed-ER and imaged live by confocal microscopy during amino acid starvation. Shown in panels A–E are examples from several such videos, where the DFCP1 omegasomes coincide with ER regions (boxed areas).