Extended Data Figure 1: Autophagy induction in Xbp1-deficient intestinal epithelial cells.
From: Paneth cells as a site of origin for intestinal inflammation
a, Xbp1 expression in shCtrl and shXbp1 MODE-K cells (n = 7/6; unpaired Student’s t-test; mean ± s.e.m.). b, c, Immunoblot of shCtrl and shXbp1 MODE-K cells. d, Immunoblot after autophagosome–lysosome fusion inhibition via bafilomycin in silenced MODE-K intestinal epithelial cells. e, Silenced MODE-K intestinal epithelial cells after GFP–LC3 reporter transfection (scale bars, 5 μm) with green punctae per cell quantification in f (n = 14; unpaired Student’s t-test; mean ± s.e.m.). g, TEM of shCtrl and shXbp1 cells. Note double-membraned structure with engulfed contents characteristic of autophagosomes (white arrows; n = 10). Scale bars, 0.5 μm. h, Quantification of occupied area and average size of autophagic vacuoles from g (n = 10; unpaired Student’s t-test; mean ± s.e.m.). i, j, Densitometry of Fig. 1a (i; n = 5/4; unpaired Student’s t-test; mean ± s.e.m.) and Fig. 1b (j; n = 3; unpaired Student’s t-test; mean ± s.e.m.). k, Low-magnification (×1,380 , original magnification here and in the remainder of this legend) TEM image of Paneth cells from wild-type mice (1), demonstrating the abundant endoplasmic reticulum (ER) and characteristic secretory granules at the apical, lumenally (L) oriented side. (2) Higher magnification (×5,520) of inset ‘2’ in (1) demonstrating typical secretory granules in Paneth cells from wild-type mice. (3) High magnification (×20,700) of inset ‘3’ in (1) illustrating a double membrane structure characteristic of an autophagosome (white arrow) in close proximity to the ER and a mitochondrion. (4) Low magnification (×2,160) TEM image of Paneth cell remnants present in Xbp1ΔIEC small intestinal crypts, which lack expansion of the ER and exhibit only minuscule granule remnants. (5) Higher magnification (×9,000) of inset ‘5’ in (4), demonstrating degradative autophagic vacuoles (black arrows), in close proximity to mitochondria, and the virtual absence of ER membranes. (6) High-power (×14,400) magnification of inset ‘6’ in (4), illustrating a double-membrane structure (white arrow) characteristic of autophagosomes, and a degradative autophagic vacuole (black arrow). ER, endoplasmic reticulum; L, lumen; M, mitochondrion; N, nucleus. Scale bars represent 2 μm, 0.5 μm and 200 nm, respectively. Results represent three (b, c) or two (d, k) independent experiments. *P < 0.05, ***P < 0.001.
