Figure 4. The FBXW5-mediated degradation of SEC23B limits the autophagic flux in the presence of nutrients.

(A) RPE1-hTERT cells were transfected with a non-targeting (NT) oligo or a FBXW5-targeting siRNA oligo. Forty-eight hours after transfection, cells were re-plated onto coverglass for immunofluorescence with an anti-LC3 antibody. Where indicated, cells were treated with Bafilomycin A1 (BafA1) for 4 hr before fixation. Images of endogenous LC3 puncta underwent automated processing with at least 300 cells counted per sample. Because in several images LC3·puncta were too close to be distinguished, we adopted LC3 puncta area as a criterion for our analysis. The data are presented as mean ±SD (right panel). Scale bar, 10 μm. (B) HeLa and RPE1-hTERT cells were transfected with the indicated siRNAs. Seventy-two hours after transfection, the cells were harvested for immunoblotting. (C) U-2OS cells stably expressing tandem fluorescent-tagged LC3 (pBabe-mCherry-EGFP-LC3) were transfected with a NT oligo or a FBXW5 siRNA oligo, alone or in combination with a SEC23B-targeting siRNA oligo as indicated. Forty-eight hours after transfection, cells were replated onto coverglass, followed by fixation twenty-four hours after replating. Images of mCherry-EGFP-LC3 puncta underwent automated processing with at least 100 cells counted per sample. The data are presented as mean ±SD (right panel). The yellow and red bars represent green +red double positive LC3 puncta (autophagosome) and red only positive LC3 puncta (autolysosome), respectively. Scale bar, 10 μm. (D) U-2OS cells were infected with lentiviruses expressing either wild-type SEC23B or SEC23B(S186D). Twenty-four hours after infection, cells were fixed for immunofluorescence. Images of endogenous LC3 puncta underwent automated processing with at least 300 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm. (E) U-2OS cells were infected with lentivirus expressing SEC23B(S186D). Where indicated, forty-eight hours after infection, cells were treated with BafA1 prior to harvest and immunoblotting. (F) U-2OS cells were infected with the increasing amounts of lentivirus expressing SEC23B. Forty-eight hours after infection, cells were harvested for immunoblotting. (G) RPE1-hTERT cells stably expressing GFP-tagged LC3 were transfected with a NT oligo or the indicated siRNA oligos. Forty-eight hours after transfection, cells were replated onto coverglass, followed by treatment with EBSS for 1 hr and fixation. Images of GFP-LC3 puncta underwent automated processing with at least 300 cells counted per sample. The data are presented as mean ±SD.

Figure 4—figure supplement 1. The FBXW5-mediated degradation of SEC23B limits the autophagic flux in the presence of nutrients.

(A) RPE1-hTERT cells stably expressing GFP-LC3 were transfected with either a non-targeting siRNA oligo (NT) or an siRNA oligo targeting FBXW5 RNA. Forty-eight hours after transfection, cells were replated onto coverglass. Where indicated, cells were treated with Bafilomycin A1 (BafA1) for 4 hr before fixation with cold methanol. Images of the GFP-LC3 puncta underwent automated processing with at least 300 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm. (B) RPE1-hTERT cells were transfected with the indicated siRNAs. Seventy-two hours after transfection, where indicated, cells were treated with Bafilomycin A1 (BafA1) for one hour prior to harvest for immunoblotting. (C) RPE1-hTERT cells stably expressing GFP-LC3 were transfected with either a non-targeting siRNA oligo (NT) or an siRNA oligo targeting FBXW5 RNA. Forty-eight hours after transfection, cells were replated onto coverglass for immunofluorescence with an anti-LC3 antibody. Cells were nutrient-starved with EBSS for 4 hr before fixation with cold methanol. Images of the LC3 puncta underwent automated processing with at least 300 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm. (D) U-2OS cells infected with either an empty virus (EV) or lentivirus expressing SEC23B(S186D) were transfected with the indicated siRNAs. Seventy-two hours after transfection, cells were treated with EBSS prior to harvest for immunoblotting.