Tubular ER structures shaped by ER-phagy receptors engage in stress-induced Golgi bypass (Song et al)
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Original datasets and Supplemental Videos : Tubular ER structures shaped by ER-phagy receptors engage in stress-induced Golgi bypass
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Figure 1. ER tubular structure formation during the stress-induced unconventional secretion of ΔF508-CFTR was analyzed by surface biotinylation assay (A), immunocytochemistry (B-F), CLEM analysis (G), and TIRF microscopy analysis (J). Figure 2. ER-TBs are dynamic structures associated with microtubules, as shown by EM imaging (A), immunocytochemistry (B-F) and time-lapse imaging (G, H). Figure 3. ATL3 and RTN3 are required for ER-TB formation and unconventional secretion of ΔF508-CFTR, as demonstrated by immunocytochemistry (A-D), immune-gold EM imaging (E), and surface biotinylation assay (F-I). Figure 4. ATL3 and RTN3L are involved in non-degradative autophagy, as shown by immunocytochemistry (A-F) and EATR assay (G-I). Figure 5. Combined expression of ATL3 and RTN3L induces ΔF508-CFTR UPS and ER-TB formation, as revealed by immunocytochemistry (A-C), surface biotinylation assay (D, E, I-L), EM imaging (F), and EATR assay (G, H). Figure 6. Combined expression of ATL3 and RTN3L evokes CFTR currents in cells expressing ΔF508-CFTR by whole-cell patch-clamp recording. Figure 7. ATL3 contributes to Golgi-independent trafficking of the SARS-CoV-2 S protein, as demonstrated by immunocytochemistry (A, B), EM imaging (C, D), immunoblot (E, F), qPCR (G), and plaque assay (H). Figure S1. Unconventional protein secretion of CFTR and ER-tubular body (ER-TB) formation were analyzed by immunocytochemistry (A), EM imaging (B, D, E), CLEM analysis (C), and TIRF imaging (F). Figure S2. ATL3 and RTN3 participate in ER-TB formation and the unconventional secretion of ΔF508-CFTR, whereas FAM134B and TEX264 do not, as shown by qPCR (A, B), immunoblot (C, D), immunocytochemistry (E, F), and surface biotinylation assay (G, H). Figure S3. Control experiments of the ER autophagy tandem reporter (EATR) assay. Figure S4. The GABARAP-interacting motif (GIM) of ATL3 and the LC3-interacting region (LIR) of RTN3L participate in ER-TB formation, as revealed by co-IP (A), CLEM assay (B), and immunocytochemistry (C-F). Figure S5. The short form of RTN (RTN3S) does not induce ER-TB formation or the UPS of ΔF508-CFTR, as shown by immunocytochemistry (A, B) and surface biotinylation assay (C, D). Figure S6. ATL3 is involved in the UPS of SARS-CoV-2 S protein, as shown by immunocytochemistry (A, B, G, H), immuno-gold EM imaging (C, D), and surface biotinylation assay (E, F). Figure S7. Proteomic analysis of ER fractions in cells after UPS blockade by ATL3 and RTN3 knockdown. All uncropped Western Blot images are shown in “Western Blot uncropped data”. Supplemental Video Video S1. Time-lapse imaging using TIRF. Video S2. Dynamic movement of ER-TB along the microtubules using time-lapse imaging. Video S3. Time-lapse imaging of ER-TB fusion events.