Figure 1.
CPP-induced autophagy is dependent on the conjugation system.
(A–B) Wild-type MEF were treated with CPP with or without CQ (50 µM) or BafA1 (500 nM), or were starved by incubating in EBSS as a positive control for autophagy initiation, followed by Western blotting for LC3 and p62 (A), and long-lived protein degradation assay (B). (C–E) HEK293 cells stably expressing FLAG-Atg4BC74S or an empty vector were treated with EBSS or CPP and analyzed by Western blotting (C) and quantification of GFP-LC3 puncta (D, E). (F–I) Wild-type, Atg5−/−, and Atg3−/− MEFs were transduced with adenoviral GFP-LC3. Forty-eight hours later cells were treated with EBSS or CPP. Selected Atg proteins and p62 were detected by Western blotting (F, G). GFP-LC3 puncta were quantified (H, I). Western blots shown are from one representative experiment of two to three performed for each panel. Quantitative data (mean±SEM) are from 3–5 experiments. *: P<0.05, **: P<0.01.
Figure 2.
CPP-induced autophagy is dependent on FIP200 and Beclin 1.
Wild-type and FIP200−/− MEFs (A–C), and Beclin 1 wild type and Beclin 1-low U251 cells (D–F) stably expressed GFP-LC3. They were treated with EBSS or CPP, followed by Western blotting analysis (A, D) and quantification of GFP-LC3 puncta (B, C, E, F). Asterisk in A indicates a non-specific band. Western blots shown are from one representative experiment of two to three performed for each panel. Quantitative data (mean±SEM) are from 3–5 experiments. *: P<0.05, **: P<0.01.
Figure 3.
Atg9 is inhibitory for CPP-induced autophagy.
Atg9 wild type and Atg9-deficient MEFs stably expressing GFP-LC3 were treated with EBSS or CPP followed by Western blotting analysis (A) and quantification of GFP-LC3 puncta (B–D). Scale bars: 10 µm in B. Western blots shown are from one representative experiment of two to three performed for each panel. Quantitative data (mean±SEM) are from 3–5 experiments. *: P<0.05, **: P<0.01.
Figure 4.
CPP-induced autophagy is independent of the signaling of mTOR, MAPK and ER stress.
(A–C) HEK293 cells were treated with EBSS or CPP, thapsigargin (TG, 0.5 µM, 16h) or MG132 (1 µM, 16 h), followed by Western blotting with indicated antibodies. (D) Wild-type and IRE1−/− MEFs were treated with EBSS or CPP, followed by Western blotting with indicated antibodies. Western blots shown are from one representative experiment of two to three performed for each panel.
Figure 5.
GFP-LC3-positive autophagosomes induced by CPP do not colocalize with the Golgi apparatus or the mitochondria.
(A) HEK293 cells stably expressing GFP-LC3 were treated with CPP, fixed and stained with anti-GM130 and Cy3-conjugated secondary antibody. (B) The same cell line was labeled with MitoTracker Red (20 nM) 30 min prior to CPP treatment. Images of the CPP-treated cells were acquired through z-sectioning and deconvoluted. Scale bars: 10 µm.
Figure 6.
GFP-LC3 positive autophagosomes induced by CPP interact with ER membranes.
(A) HEK293 cells stably expressing both GFP-LC3 and CFP-ER were treated with CPP. Fluorescent images were taken and CFP channel was pseudo-colored in red. (B) HEK293 cells stably expressing GFP-LC3 were treated with CPP, then fixed and stained with an anti-calnexin antibody and then Cy3-conjugated secondary antibody. (C) HEK293 cells stably expressing both GFP-LC3 and Sec61b-mCherry was treated with CPP, images were taken at different z-sections (the section is indicated) and deconvoluted. Arrows indicate the colocalization of LC3 and Sec61b signals. Constructed z-stacks are shown in movie S1. (D) A single clone of HEK293 cells stably expressing both GFP-LC3 and Sec61b-mCherry was treated with CPP and then lysed. Fluorescence images of the lysates were acquired. Vesicles showing the presence of both GFP-LC3 and Sec61b-mCherry were observed. Scale bars: 5 µm in A, C and D; 10 µm in B.
Figure 7.
GFP-LC3 positive autophagosomes induced by CPP are associated with DFCP1 signals.
(A) HEK293 cells stably expressing both GFP-LC3 and RFP-DFCP1 were treated with CPP and images of live cells were taken by confocal microscopy. (B–D) HEK293 cells stably expressing GFP-DFCP1 was treated with CPP with or without 3-MA (10 mM) (B–C), or BAPTA-AM (10 mM) (C) 30 min prior to CPP. GFP-LC3 puncta were quantified (B–C). Scale bars: 5 µm in A; 10 µm in B. Quantitative data (mean±SEM) are from 3–5 experiments. *: P<0.05, **: P<001.