Figure 5.
Spatial assessment of ER-phagy activity and ER architecture in different tissues. (A) Co-labeling with LAMP1 indicates that almost all red-positive (RFP+) compartments are also lysosomal marker positive and yield purple (red: RFP+, blue: LAMP1+) signals in the hepatocytes of 8-wk ER-TRG (+/−) mice. Lysosomes not engaged in autophagy (LAMP1 negative) remain blue. Inset: enlarged image of area indicated by dashed frame. Scale bar: 20 μm. (B) Quantitative analysis of different regions of liver from ER-TRG (+/−) reporter mice at adulthood. The calculations were performed for regions inside or outside the yellow dashed box in mouse liver (S3A), referred to as the inner and outer regions. The inner regions represent the vicinity of the CV zone, while the outer regions represent the areas away from the CV zone. The pixel number of RFP-only signals and total RFP signals were calculated by ImageJ and the ratio of RFP: total area (RFP+GFP) was quantified (left). The intensity of RFP-only signals and total RFP signals were calculated by ImageJ on a pixel-by-pixel basis and the fluorescence ratio of RFP: total RFP was quantified (right). Values were stated as mean ± SEM (n = 6). Statistical differences were evaluated using Student’s t test. ***P < 0.001. (C) Quantification of RFP-only ER-lysosomes (RFP+LAMP1+) in liver sections obtained from ER-TRG mice. Values were stated as mean ± SD (n = 4). (D–F) Representative images and quantitative analysis of ER-TRG signals in the liver of control and Atg7-knockdown mice. 8-wk-old ER-TRG (+/−) mice were injected with AAV-shNC and AAV-shAtg7 and underwent a 4-wk infection period. Quantitative analysis of RFP+ signals and immunoblotting of RFP cleavage indicated reduced ER-phagy in hepatocytes after Atg7 knockdown (F). The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. The efficiency of ATG7 knockdown is evidenced by the expression levels of ATG7, p62, and LC3 in liver samples. GAPDH and Tubulin served as internal controls. Values were stated as mean ± SEM (n = 4 per group). Statistical differences were analyzed using Student’s t test. *P < 0.05, ***P < 0.001. Scale bar: 20 μm. (G–J) Representative confocal images depicting ER-phagy levels in the lung and adipose tissues of ER-TRG (+/−) mice. Quantitative analysis including alveoli and bronchioles (G), brown adipose tissue (BAT), and epididymal white adipose tissue (eWAT) (I) indicated different ER abundance and positioning of ER-phagy signals within tissues (n = 4–6). Magnified images highlight RFP-only signals in cells of various tissues. Adult male ER-TRG (+/−) mice were used in all experiments. Values were stated as mean ± SEM (n = 6) (H). Values were stated as mean ± SD (n = 4–5) (J). Statistical differences were analyzed using one-way ANOVA. **P < 0.01, ***P < 0.001. Scale bar: 20 μm. (K) Representative confocal images depicting ER-phagy levels in the heart sections and quantitative analysis of ER-phagy level of heart in ER-TRG (+/−) mice. Values were stated as mean ± SD (n = 5). Scale bar: 20 μm. (L) Immunoblot analysis of ssRFP-GFP-KDEL expression in various tissues of transgenic mice. Tissue homogenates from one wild-type and one ER-TRG (+/−) transgenic mouse were probed with specific antibodies. Both full-length RFP-GFP and RFP fragments were detected using an anti-RFP antibody. The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. GAPDH and Tubulin were used as internal controls. See also Fig. S3. Source data are available for this figure: SourceData F5.

Spatial assessment of ER-phagy activity and ER architecture in different tissues. (A) Co-labeling with LAMP1 indicates that almost all red-positive (RFP+) compartments are also lysosomal marker positive and yield purple (red: RFP+, blue: LAMP1+) signals in the hepatocytes of 8-wk ER-TRG (+/−) mice. Lysosomes not engaged in autophagy (LAMP1 negative) remain blue. Inset: enlarged image of area indicated by dashed frame. Scale bar: 20 μm. (B) Quantitative analysis of different regions of liver from ER-TRG (+/−) reporter mice at adulthood. The calculations were performed for regions inside or outside the yellow dashed box in mouse liver (S3A), referred to as the inner and outer regions. The inner regions represent the vicinity of the CV zone, while the outer regions represent the areas away from the CV zone. The pixel number of RFP-only signals and total RFP signals were calculated by ImageJ and the ratio of RFP: total area (RFP+GFP) was quantified (left). The intensity of RFP-only signals and total RFP signals were calculated by ImageJ on a pixel-by-pixel basis and the fluorescence ratio of RFP: total RFP was quantified (right). Values were stated as mean ± SEM (n = 6). Statistical differences were evaluated using Student’s t test. ***P < 0.001. (C) Quantification of RFP-only ER-lysosomes (RFP+LAMP1+) in liver sections obtained from ER-TRG mice. Values were stated as mean ± SD (n = 4). (D–F) Representative images and quantitative analysis of ER-TRG signals in the liver of control and Atg7-knockdown mice. 8-wk-old ER-TRG (+/−) mice were injected with AAV-shNC and AAV-shAtg7 and underwent a 4-wk infection period. Quantitative analysis of RFP+ signals and immunoblotting of RFP cleavage indicated reduced ER-phagy in hepatocytes after Atg7 knockdown (F). The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. The efficiency of ATG7 knockdown is evidenced by the expression levels of ATG7, p62, and LC3 in liver samples. GAPDH and Tubulin served as internal controls. Values were stated as mean ± SEM (n = 4 per group). Statistical differences were analyzed using Student’s t test. *P < 0.05, ***P < 0.001. Scale bar: 20 μm. (G–J) Representative confocal images depicting ER-phagy levels in the lung and adipose tissues of ER-TRG (+/−) mice. Quantitative analysis including alveoli and bronchioles (G), brown adipose tissue (BAT), and epididymal white adipose tissue (eWAT) (I) indicated different ER abundance and positioning of ER-phagy signals within tissues (n = 4–6). Magnified images highlight RFP-only signals in cells of various tissues. Adult male ER-TRG (+/−) mice were used in all experiments. Values were stated as mean ± SEM (n = 6) (H). Values were stated as mean ± SD (n = 4–5) (J). Statistical differences were analyzed using one-way ANOVA. **P < 0.01, ***P < 0.001. Scale bar: 20 μm. (K) Representative confocal images depicting ER-phagy levels in the heart sections and quantitative analysis of ER-phagy level of heart in ER-TRG (+/−) mice. Values were stated as mean ± SD (n = 5). Scale bar: 20 μm. (L) Immunoblot analysis of ssRFP-GFP-KDEL expression in various tissues of transgenic mice. Tissue homogenates from one wild-type and one ER-TRG (+/−) transgenic mouse were probed with specific antibodies. Both full-length RFP-GFP and RFP fragments were detected using an anti-RFP antibody. The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. GAPDH and Tubulin were used as internal controls. See also Fig. S3. Source data are available for this figure: SourceData F5.

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