Fig. 1 

ISX induces ER-phagy (A) Magnified TEM pictures showing ER. Panc1 cells were treated for 48h with DMSO, 20 µM ISX, and 1.0 µM Gem. Arrows indicate normal (A1) or dilated (A2, A3) ER. (B) Quantification of ER stress by measuring the number of expanded ER per Panc1 cell in each treatment group as shown in (A). I/G=ISX/Gem. Each dot represents one cell (mean ± SD). (C) The area of autophago- and autolysosomes per cell. Each dot represents one cell (mean ± SD). (D) Magnified TEM pictures showing autophagosomes (D1) and autolysosomes (D2, D3) in double-treated cells (ISX 20 µM, Gem 1.0 µM for 48h). (E) Number of autophago- and autolysosomes per Panc1 cell. Each dot represents one cell (mean ± SD). Treatment as in (A). (F) Immunofluorescence pictures of Panc1 cells transiently transfected with ER-Keima reporter and treated as described in (A). The pictures shown are overlay images depicting non-phagocytosed ER (green) and ER-derived autolysosomes (red). Scale bar: 60µm. Shown is one representative experiment of n=2. (G, H) Flow cytometric quantification of the ER-phagy in Panc1 cells stably expressing ER-Keima reporter, treated as described in (A) (mean of n=3 ± SD) and histogram of flow cytometry. (I) Relative mRNA expression of ER-phagy receptors (FAM134B, CCPG1, SEC62, RTN3, TEX264) in Panc1 cells treated for 48h with DMSO or 20 µM ISX (mean of n=3 ± SD). (J) Western blot analysis of FAM134B levels in Panc1 cells treated for 48h as described in (A). β-Actin was used as a loading control. One representative of n=3 is shown. (K) Quantification of FAM134B protein levels as depicted in (J) (mean of n=3 ± SD). (L) Colocalization of mCherry-LC3B (red) and endogenous FAM134B (green) of Panc1 cells treated for 48h. Treatment as in (A). One representative of n=4 is shown. Scale bar: 50µm in the larger panel and 10µm in the magnified insets