Figure S3.
Spatial assessment of ER-phagy activity and ER architecture in different tissues (related to Fig. 5). (A) Colabeling with CTSD indicates that almost all red-positive compartments are also lysosomal marker positive and yield purple (red: RFP+, blue: CTSD+) signals in the hepatocytes of 8-wk-old ER-TRG (+/−) mice. Lysosomes not engaged in autophagy (CTSD negative) remain blue. An inset shows an enlarged view of the area indicated by the yellow dashed frame. The area inside the yellow dashed box represents the vicinity of the CV zone, while the area outside the yellow dashed box represents the areas away from the CV zone. Scale bar: 20 μm. (B) Quantification of RFP-only ERlysosomes (RFP+CTSD+) in liver sections obtained from ER-TRG mice. Values were stated as mean ± SD (n = 4). (C and D) Immunoblotting analysis of ATG7, p62, and LC3 in primary hepatocytes indicates the efficiency of ATG7 knockdown by AAV infection. Cell extracts from AAV-shNC and AAV-shAtg7 transgenic mice were examined via immunoblotting with specific antibodies. GAPDH and Tubulin served as internal controls. Values were stated as mean ± SEM (n = 5 per group). Statistical significance was determined using Student’s t test, with ***P < 0.001. (E–G) Confocal imaging and quantitative analysis of the intestines and colons from adult ER-TRG (+/−) reporter mice reveal varied ER abundance and ER-phagy activity in the villi of the small intestine and crypt (left) (E and F), compared to the comparable ER-phagy activity in colon cells (right) (E and G). Values were stated as mean ± SEM (n = 4) (F). Values were stated as mean ± SD (n = 4) (G). Statistical differences were analyzed using one-way ANOVA. *P < 0.05, **P < 0.01. Scale bar: 20 μm. (H and I) ER abundance and positioning of ER-phagy signals within the mouse brain. Magnified images highlight RFP-only signals in cells of various tissues, with specific focus on an astrocyte (#1), neurons around the hippocampus (#2), and neurons around the cortex (#3) in the brain. Quantitative analysis of ER-phagy puncta of cells in the brain of adult male ER-TRG (+/−) mice (I). n = 60–80 cells per group from three mice. Scale bars: 200 μm (thumbnail), 10 μm (inset). (J and K) Confocal imaging and quantitative analysis of ER-phagy level of the testis in adult male ER-TRG (+/−) reporter mice. Values were stated as mean ± SD (n = 4). Scale bar: 20 μm. (L–O) Confocal imaging of the adrenal glands and epididymis from adult ER-TRG (+/−) mice. Magnified images depicting the smooth ER (SER) architecture and localization of ER-phagy signals within the adrenal glands (L and N). The zona grannulosa (ZG), zona fasciculata (ZF), and zona reticulosa (ZR) regions were examined. A magnified view highlights RFP-only signals in cells of adrenal glands (n = 4). Scale bar: 10 μm. Magnified images and quantitative analysis show RFP-only signals in cells of epididymis, with the initial segment (IS), caput, and cauda regions displaying varying levels of ER-phagy across different cell types (n = 4) (M and O). Values were stated as mean ± SEM. Statistical differences were analyzed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 20 μm. Source data are available for this figure: SourceData FS3.

Spatial assessment of ER-phagy activity and ER architecture in different tissues (related to Fig. 5 ). (A) Colabeling with CTSD indicates that almost all red-positive compartments are also lysosomal marker positive and yield purple (red: RFP+, blue: CTSD+) signals in the hepatocytes of 8-wk-old ER-TRG (+/−) mice. Lysosomes not engaged in autophagy (CTSD negative) remain blue. An inset shows an enlarged view of the area indicated by the yellow dashed frame. The area inside the yellow dashed box represents the vicinity of the CV zone, while the area outside the yellow dashed box represents the areas away from the CV zone. Scale bar: 20 μm. (B) Quantification of RFP-only ERlysosomes (RFP+CTSD+) in liver sections obtained from ER-TRG mice. Values were stated as mean ± SD (n = 4). (C and D) Immunoblotting analysis of ATG7, p62, and LC3 in primary hepatocytes indicates the efficiency of ATG7 knockdown by AAV infection. Cell extracts from AAV-shNC and AAV-shAtg7 transgenic mice were examined via immunoblotting with specific antibodies. GAPDH and Tubulin served as internal controls. Values were stated as mean ± SEM (n = 5 per group). Statistical significance was determined using Student’s t test, with ***P < 0.001. (E–G) Confocal imaging and quantitative analysis of the intestines and colons from adult ER-TRG (+/−) reporter mice reveal varied ER abundance and ER-phagy activity in the villi of the small intestine and crypt (left) (E and F), compared to the comparable ER-phagy activity in colon cells (right) (E and G). Values were stated as mean ± SEM (n = 4) (F). Values were stated as mean ± SD (n = 4) (G). Statistical differences were analyzed using one-way ANOVA. *P < 0.05, **P < 0.01. Scale bar: 20 μm. (H and I) ER abundance and positioning of ER-phagy signals within the mouse brain. Magnified images highlight RFP-only signals in cells of various tissues, with specific focus on an astrocyte (#1), neurons around the hippocampus (#2), and neurons around the cortex (#3) in the brain. Quantitative analysis of ER-phagy puncta of cells in the brain of adult male ER-TRG (+/−) mice (I). n = 60–80 cells per group from three mice. Scale bars: 200 μm (thumbnail), 10 μm (inset). (J and K) Confocal imaging and quantitative analysis of ER-phagy level of the testis in adult male ER-TRG (+/−) reporter mice. Values were stated as mean ± SD (n = 4). Scale bar: 20 μm. (L–O) Confocal imaging of the adrenal glands and epididymis from adult ER-TRG (+/−) mice. Magnified images depicting the smooth ER (SER) architecture and localization of ER-phagy signals within the adrenal glands (L and N). The zona grannulosa (ZG), zona fasciculata (ZF), and zona reticulosa (ZR) regions were examined. A magnified view highlights RFP-only signals in cells of adrenal glands (n = 4). Scale bar: 10 μm. Magnified images and quantitative analysis show RFP-only signals in cells of epididymis, with the initial segment (IS), caput, and cauda regions displaying varying levels of ER-phagy across different cell types (n = 4) (M and O). Values were stated as mean ± SEM. Statistical differences were analyzed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 20 μm. Source data are available for this figure: SourceData FS3.

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