Generation of GFP–LC3–RFP knock-in mice and the detection of starvation-induced autophagy.

(A) Schematic illustration of the knock-in strategy to generate R26-STOP-GLR mice expressing the GFP–LC3–RFP autophagic flux reporter. The cassette containing the CAG promoter, a loxP-flanked STOP cassette, and the GFP–LC3–RFP reporter was inserted into the intron between exons 1 and 2 of the Rosa26 locus. (B) Schematic illustration of the principle of the GFP–LC3–RFP reporter to evaluate autophagic flux. (C, E, G) Representative fluorescence and corresponding GFP/RFP ratio images of the liver (C), skeletal muscle (E), and brain (G). Mice (3–4-month-old) were starved for 24 h or 48 h. Scale bars, 1 mm (low magnification) and 200 µm (high magnification). (D, F, H) Quantification of the GFP/RFP ratio in the liver (D), skeletal muscle (F), and brain (H). Each dot represents an individual mouse; bars indicate mean values; n = 9–10 per group. Differences among groups were analyzed by one-way ANOVA with Tukey’s post hoc test. This figure was created using BioRender.com.

A simple, rapid, and semi-high-throughput measurement of the GFP/RFP ratio using a microplate reader.

(A) Schematic illustration of the microplate reader-based assay detecting GFP–LC3–RFP reporter signals. (B) Soluble fractions from liver and brain lysates of WT and R26-GLR mice (3–4-month-old) were serially diluted and measured using a microplate reader (n = 3 per group). Data are presented as mean ± SD values. The coefficients of determination (R²) from linear regressions of measurements of tissue concentration and GFP signal for R26-GLR mice are shown. (C) Soluble fractions from tissue lysates of WT and R26-GLR mice (3–4-month-old) were diluted 4× and measured using a microplate reader (n = 3 per group). Bars represent mean ± SD values. (D) GFP/RFP ratios in the liver, skeletal muscle, and brain under 24- or 48-h starvation conditions, calculated from microplate reader measurements. Each dot represents an individual mouse; bars indicate mean values; n = 6–9 per group. Differences among groups were analyzed by one-way ANOVA with Tukey’s post hoc test. This figure was created using BioRender.com.

Uniform basal autophagic flux across multiple tissues during embryonic development.

(A) Schematic illustration of the breeding strategy for obtaining GFP–LC3–RFP–expressing Atg5 knockout embryos (E19.5). Embryo dissection and detection were performed as described. (B) GFP/RFP ratios in Atg5 wild-type (WT), heterozygous (HT), or knockout (KO) embryos measured using a microplate reader. Each dot represents an individual embryo; bars indicate mean values; n = 4–5 per group. Differences among groups were analyzed by one-way ANOVA with Tukey’s post hoc test. (C) Representative GFP/RFP ratio images of the body of Atg5 WT and Atg5 KO embryos. Scale bar, 1 mm; Mu, skeletal muscle; He, heart; Lu, lung; Li, liver; Pa, pancreas; Ki, kidney; and In, intestine. (D) Representative fluorescence and corresponding GFP/RFP ratio images of major tissues of Atg5 WT and Atg5 KO embryos. Scale bar, 100 µm. (E) The GFP/RFP ratio calculated from tissue imaging of Atg5 WT, Atg5 HT, and Atg5 KO embryos. Each dot represents an individual embryo; bars indicate mean values; n = 4–6 per group. Differences among groups were analyzed by one-way ANOVA with Tukey’s post hoc test. This figure was created using BioRender.com.

Distinct levels of basal autophagic flux between adult tissues.

(A) Experimental design for tamoxifen-inducible Atg5 KO mice. Mice (3–4-month-old) were administered tamoxifen once per week for four weeks. One month after the last tamoxifen injection, mice were sacrificed for analysis. (B) Representative immunoblotting of the brain and kidney from R26-GLRKI/+;Atg5+/+, R26-GLRKI/+;Atg5flox/flox, R26-GLRKI/+;Atg5flox/+;CreERT2Tg/+, and R26-GLRKI/+;Atg5flox/flox;CreERT2Tg/+ mice with or without tamoxifen administration. (C-E) Representative fluorescence and corresponding GFP/RFP ratio images of the liver (C), kidney (D), and brain (E) of R26-GLRKI/+;Atg5F/+;CreERT2Tg/+and R26-GLRKI/+;Atg5F/F;CreERT2Tg/+mice. Scale bar, 1 mm. (F) The GFP/RFP ratio calculated from tissue imaging of the liver, kidney, and brain of R26-GLRKI/+;Atg5F/+;CreERT2Tg/+ and R26-GLRKI/+;Atg5F/F;CreERT2Tg/+ mice. Each dot represents an individual mouse; bars indicate mean values; n = 7–15 per group. Differences between groups were analyzed by Welch’s t-test. This figure was created using BioRender.com.

Distinct levels of basal autophagic flux in adult tissues and embryos.

(A) Tissue-specific GFP/RFP ratios, as measured by microplate reader, of R26-GLRKI/+;Atg5F/F;CreERT2Tg/+mice, normalized to corresponding values of R26-GLRKI/+;Atg5F/+;CreERT2Tg/+mice (set as 100%). Each dot represents an individual mouse; bars indicate mean ± SD values; n = 10–11 per group. (B) The GFP/RFP ratios of various tissues from Atg5 KO embryos and adult mice, normalized to the corresponding values in Atg5 WT controls (set as 100%). The data of embryos and adults are from Fig. 3E and Fig. 6A, respectively. This figure was created using BioRender.com.

Comparable levels of basal autophagic flux among distinct neuronal populations.

(A–C) Representative fluorescence and corresponding GFP/RFP ratio images of the cerebral cortex (A), hippocampus (B), and cerebellum (C) of R26-GLRKI/+;Atg5F/+;CreERT2Tg/+ and R26-GLRKI/+;Atg5F/F;CreERT2Tg/+ mice. Yellow arrows indicate Hoechst+/NeuN+ neurons, while red arrowheads indicate Hoechst+/NeuN− non-neuronal cells (B). Scale bars, 500 µm (low magnification) and 20 µm (high magnification). CA, Cornu Ammonis; DG, dentate gyrus. (D) The GFP/RFP ratio of neurons in the cortex and hippocampus and cerebellar Purkinje cells of R26-GLRKI/+;Atg5F/+;CreERT2Tg/+and R26-GLRKI/+;Atg5F/F;CreERT2Tg/+ mice. Each dot represents an individual mouse (30 neurons were quantified per brain region for each mouse); bars indicate mean values; n = 7 per group. Differences between groups were analyzed by Welch’s t-test.