Figure 9.
Assessment of ER-phagy activity in different tissues in inducible ER-TRG mice. (A) Immunoblot analysis showing the expression of ssRFP-GFP-KDEL in tissues isolated from inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days and the Rosa CreERT2−/−; LSL-RFP-GFP-KDEL+/−was used as control. Full-length RFP-GFP, double red asterisk (**) and RFP fragments, single red asterisks (*), indicative of ER-phagy activity, were detected using an anti-RFP antibody. GAPDH and Tubulin were used as internal controls. (B) Quantification of immunoblots in panel A. The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. The Rosa CreERT2−/−; LSL-RFP-GFP-KDEL+/−was used as control. Values were stated as mean ± SEM (n = 3–4 per group). Statistical differences were evaluated using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (C and D) ER-phagy levels and quantitative analysis in the pancreas of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. 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 were quantified (right). Values were stated as mean ± SEM (n = 4 per group). Statistical differences were evaluated using Student’s t test. *P < 0.05, **P < 0.01. Scale bar: 20 μm. (E and F) ER-phagy levels and quantitative analysis in the liver of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. Values were stated as mean ± SEM (n = 4 group). Statistical differences were evaluated using Student’s t test. **P < 0.01. Scale bar: 20 μm. (G and H) ER-phagy levels and quantitative analysis in the small intestine of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. The mice were induced by tamoxifen for 2 or 3 days and the samples were analyzed after 5 or 7 days. Values were stated as mean ± SEM (n = 3–5 per group). Statistical differences were evaluated using two-way ANOVA. ###P < 0.001 (comparison between different cells under the same treatment), *P < 0.05 (comparison between the same cell under different treatments). Scale bar: 20 μm. (I and J) ER-phagy levels and quantitative analysis in the Purkinje cells of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. The images were enlargement of Fig. S5 A. The number of RFP-only puncta per soma of Purkinje cells were calculated by ImageJ and quantified in each treatment. Values were stated as mean ± SEM (n = 4 per group). Statistical differences were evaluated using Student’s t test. *P < 0.05. Scale bar: 20 μm. See also Fig. S5. Source data are available for this figure: SourceData F9.

Assessment of ER-phagy activity in different tissues in inducible ER-TRG mice. (A) Immunoblot analysis showing the expression of ssRFP-GFP-KDEL in tissues isolated from inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days and the Rosa CreERT2−/−; LSL-RFP-GFP-KDEL+/−was used as control. Full-length RFP-GFP, double red asterisk (**) and RFP fragments, single red asterisks (*), indicative of ER-phagy activity, were detected using an anti-RFP antibody. GAPDH and Tubulin were used as internal controls. (B) Quantification of immunoblots in panel A. The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. The Rosa CreERT2−/−; LSL-RFP-GFP-KDEL+/−was used as control. Values were stated as mean ± SEM (n = 3–4 per group). Statistical differences were evaluated using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (C and D) ER-phagy levels and quantitative analysis in the pancreas of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. 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 were quantified (right). Values were stated as mean ± SEM (n = 4 per group). Statistical differences were evaluated using Student’s t test. *P < 0.05, **P < 0.01. Scale bar: 20 μm. (E and F) ER-phagy levels and quantitative analysis in the liver of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. Values were stated as mean ± SEM (n = 4 group). Statistical differences were evaluated using Student’s t test. **P < 0.01. Scale bar: 20 μm. (G and H) ER-phagy levels and quantitative analysis in the small intestine of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. The mice were induced by tamoxifen for 2 or 3 days and the samples were analyzed after 5 or 7 days. Values were stated as mean ± SEM (n = 3–5 per group). Statistical differences were evaluated using two-way ANOVA. ###P < 0.001 (comparison between different cells under the same treatment), *P < 0.05 (comparison between the same cell under different treatments). Scale bar: 20 μm. (I and J) ER-phagy levels and quantitative analysis in the Purkinje cells of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. The images were enlargement of Fig. S5 A. The number of RFP-only puncta per soma of Purkinje cells were calculated by ImageJ and quantified in each treatment. Values were stated as mean ± SEM (n = 4 per group). Statistical differences were evaluated using Student’s t test. *P < 0.05. Scale bar: 20 μm. See also Fig. S5. Source data are available for this figure: SourceData F9.

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