1. An Autophagic Flux Probe that Releases an Internal Control
Takeshi Kaizuka, Hideaki Morishita, Yutaro Hama, Satoshi Tsukamoto, Takahide Matsui, Yuichiro Toyota, Akihiko Kodama, Tomoaki Ishihara, Tohru Mizushima, Noboru Mizushima 
Molecular Cell 
Volume 64, Issue 4, Pages (November 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 835-849DOI: (10.1016/j.molcel.2016.09.037)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Development of GFP-LC3-RFP-LC3ΔG, a Novel Autophagic Flux Probe
(A) Schematic representations of the measurement of autophagic flux using GFP-LC3-RFP-LC3ΔG.
(B) GFP-LC3-RFP-LC3ΔG is completely cleaved depending on the glycine at the end of the first LC3. Lysates of MEFs stably expressing GFP-LC3-RFP-LC3ΔG or GFP-LC3ΔG-RFP-LC3ΔG were analyzed by immunoblotting.
(C–E) GFP-LC3, but not RFP-LC3ΔG, was degraded in a starvation- and ATG5-dependent manner. Wild-type and Atg5−/− MEFs stably expressing GFP-LC3-RFP-LC3ΔG were starved (depletion of both serum and amino acids) for the indicated times and analyzed by immunoblotting (C), flow cytometry (D), and fluorescence microscopy (E). Histograms of fluorescence intensity versus cell count and the GFP/RFP fluorescence ratio (as a percentage of the mean of cells cultured in regular medium) are shown (n = 3) (D). Data represent mean ± SEM. The same histogram (gray) of probe (−) Atg5+/+ cells is shown in both Atg5+/+ and Atg5−/− panels for comparison. In the ratiometric images, the blue signal represents high GFP/RFP and low autophagic flux, and the red signal represents low GFP/RFP and high autophagic flux. The indicated regions are magnified in the insets (E). Scale bar, 100 μm and 5 μm (insets) (E).
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Measurement of Autophagic Flux Using a Microplate Reader
(A) Detection of starvation-induced autophagy using a microplate reader. HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG were cultured in medium containing the indicated concentration of fetal bovine serum and amino acids for the indicated times (n = 4). Data represent mean ± SEM. The GFP/RFP fluorescence ratio is expressed as a percentage relative to that at time 0. Data are representative of at least three independent experiments.
(B) Re-evaluation of the effects of autophagy inducers (blue), inhibitors (red), and inducers/inhibitors (gray) using a microplate reader. HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG were cultured in regular or starvation medium for 6, 12, or 24 hr with the indicated drugs. The GFP/RFP fluorescence ratio is expressed as a percentage relative to that of non-treated cells (n = 3). Data represent mean ± SEM. 2-DG, 2-deoxy-D-glucose; 3-MA, 3-methyladenine; AICAR, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide; BafA1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CQ, chloroquine; DFO, deferoxamine; L-NAME, Nω-Nitro-L-arginine methyl ester hydrochloride.
See also Figure S2 and Table S1.
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5. Figure 3 Screen of an Approved Drug Library for Autophagy Modulators
(A) Measurement of autophagic flux using a microplate reader. GFP-LC3-RFP-LC3ΔG-expressing HeLa cells were cultured in regular (10% serum and 1× amino acids) or starvation medium (1% serum and 0.1× amino acids) containing each drug from an approved drug library for 24 hr. The GFP/RFP fluorescence ratio is expressed as a percentage relative to that of non-treated cells (n = 3). Data represent mean ± SEM.
(B–D) Classification of the drugs identified as autophagy inducers and inhibitors based on the previously reported effect on autophagy (B), LC3 puncta formation activity (C), and function (D).
(E) The effects of representative drugs on the formation of GFP-LC3 puncta. HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG cultured in regular or starvation medium containing the indicated drugs for 2 or 24 hr. Magnified images of the indicated regions are shown in the insets. Scale bars, 10 μm (white) and 2 μm (yellow).
See also Tables S2, S3, S4, and S5.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Temporal Analysis of Induced Autophagy In Vivo in Live Zebrafish
(A) Representative images of GFP and RFP fluorescence signals in zebrafish embryos at 2 days post-fertilization (dpf) injected with GFP-LC3-RFP-LC3ΔG mRNA. Scale bar, 0.5 mm.
(B) Immunoblot analysis detecting LC3-fusion proteins in wild-type, GFP-LC3-RFP-LC3ΔG transgenic, and GFP-LC3-RFP-LC3ΔG mRNA-injected zebrafish at 2 dpf. The asterisk indicates a non-specific band.
(C) Representative fluorescence ratio images of skeletal muscle in GFP-LC3-RFP-LC3ΔG transgenic zebrafish. Zebrafish (1 dpf) were treated with 250 nM Torin1 for the indicated incubation times. Magnified images of the indicated regions are shown in the insets. Scale bar, 10 μm and 2 μm (inset). The graph shows the GFP/RFP fluorescence ratio as a percentage relative to that in untreated zebrafish at 0 hr.
(D) A schematic diagram of a fertilized egg at the one-cell stage and GFP-LC3 signals of GFP-LC3-RFP-LC3ΔG transgenic zebrafish eggs. mpf, min post-fertilization. Magnified images of the indicated regions are shown in the insets. Scale bar, 5 μm (insets).
(E) GFP-LC3 and LysoTrackerRed signals at 2 hr post-fertilization (hpf) in GFP-LC3-RFP-LC3ΔG transgenic zebrafish eggs immersed in LysoTracker Red for 2 hr. Black lines in the differential interference contrast (DIC) image indicate cell boundaries. As RFP-LC3ΔG signals were not detected as dot-like patterns (data not shown), they can be distinguished from the punctate LysoTracker Red signals. The indicated regions are shown and magnified in the insets. Scale bar, 5 μm (insets).
(F) Representative time-lapse images of fertilized eggs of GFP-LC3-RFP-LC3ΔG transgenic zebrafish at the indicated times (hpf). Scale bar, 200 μm. The graph shows the GFP/RFP fluorescence ratio in the blastomere as a percentage relative to that at 0.5 hpf.
Data are representatives of three independent experiments (A–F).
See also Figure S3 and Movie S1.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Spatial Analysis of Basal Autophagic Flux In Vivo in Live Zebrafish
(A and B) A schematic diagram of 1 dpf zebrafish and representative images of GFP and RFP fluorescence signals and DIC in the eye (lens and retina) (A) and tail (skeletal muscle and spinal cord) (B) of FIP200+/+ and FIP200−/− zebrafish embryos injected with GFP-LC3-RFP-LC3ΔG mRNA. Images of the indicated regions are shown in the insets. Scale bar, 10 μm.
(C) The graph shows the GFP/RFP fluorescence ratio of the indicated tissues of control (FIP200+/+, FIP200+/−, n = 3) and FIP200−/− (n = 4) zebrafish embryos injected with GFP-LC3-RFP-LC3ΔG mRNA. Values are expressed as a percentage relative to that of FIP200−/− zebrafish lens. Data represent mean ± SEM. ∗p < 0.05 and ∗∗p < Data are representatives of three independent experiments.
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8. Figure 6 Evaluation of Autophagic Flux in Mice
(A) Fertilized mouse embryos injected with GFP-LC3-RFP-LC3ΔG mRNA. Time (h) after fertilization is indicated. Arrowhead indicates a developmentally arrested embryo. Scale bar, 200 μm. The graph shows quantification of fluorescence intensity of normally developing embryos. Values are expressed as a percentage of the mean of embryos/eggs at the first time point. Data represent mean ± SEM (n ≥ 15).
(B) Representative fluorescence ratio images of gastrocnemius muscles of GFP-LC3-RFP-LC3ΔG transgenic mice starved for 0, 24, and 48 hr and wild-type mice. Scale bar, 200 μm. The graph shows quantification of fluorescence intensities as a percentage of the mean of that at time 0 (n = 3). Data represent mean ± SEM.
(C) Immunoblot analysis detecting GFP-LC3 turnover. Tissue homogenates were prepared from gastrocnemius muscles of two GFP-LC3-RFP-LC3ΔG transgenic mice starved for 0, 24, and 48 hr.
(D) Gastrocnemius muscles of GFP-LC3-RFP-LC3ΔG transgenic mice. To identify fiber types, we stained a serial section using antibodies against myosin heavy-chain type I and IIa. Magnified images of the indicated regions are shown in the bottom panels. The graph shows the GFP/RFP fluorescence ratio in each type of fiber (n = 10) as a percentage relative to that in type I fibers. Data represent mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 (ANOVA followed by Tukey post hoc test). Scale bars, 200 μm (white) and 40 μm (yellow).
See also Figure S4 and Movie S2.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Evaluation of Autophagic Flux Using GFP-LC3-RFP
(A) Schematic representation of the measurement of autophagic flux using GFP-LC3-RFP.
(B) Immunoblot analysis detecting cleavage efficiency of GFP-LC3-RFP. Lysates of wild-type MEFs stably expressing GFP-LC3-RFP or GFP-LC3ΔG-RFP cultured in regular medium were analyzed.
(C and D) Wild-type and Atg5−/− MEFs stably expressing GFP-LC3-RFP (bulk transformants) cultured in starvation medium for the indicated times (C) or 4 hr (D). Cell lysates were subjected to immunoblot analysis (C). The GFP and RFP fluorescence was measured by flow cytometry (D). The GFP/RFP fluorescence ratios are expressed as a percentage of that of cells cultured in regular medium (n = 3). Data represent mean ± SEM.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions