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. 2019 Aug 1;33(15-16):1083–1094. doi: 10.1101/gad.326868.119

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© 2019 Sun et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 1. — SHP expression in pancreatic acinar cells is induced by ER stress in an IRE1α–XBP1s-dependent manner. (A) Genomic tagging of endogenous Shp gene loci with 3XFlag in the 3XFlag-Shp knock-in (KI) mice (top panel) and Western blot analysis of SHP expression in different tissues (bottom panel). (Panc) Pancreas; (WAT) white adipose tissue; (BAT) brown adipose tissue; (WT) wild-type mice for negative control; (βactin) a loading control. (B) Quantitative PCR (qPCR) analysis of pancreatic Shp mRNA levels in WT mice treated with dithiothreitol (DTT) at 0.75 mmol per kilogram of body weight for 6 h. (C,D) qPCR analysis of Shp mRNA levels in AR42J acinar cells (C) or primary acinar cells (D) treated with vehicle (Veh), 100 nM thapsigargin (Tg), or 2 mM DTT for 4 h. (E,F) qPCR analysis of mRNA levels of Shp (E) and Erdj4 (F) in AR42J cells treated with vehicle, 30 µM IRE1α inhibitor 4µ8C, and/or 2 mM DTT for 4 h. (G,H) qPCR analysis of mRNA levels of Shp (G) and Erdj4 (H) in Xbp1 knockout AR42J cells (KO-1 and KO-2) or control cells (CON) with a nonspecific targeting guide, treated with vehicle or 2 mM DTT for 4 h. (I) Alignment of the human, rat, and mouse Shp promoters highlighting the putative XBP1s-binding site (underlined). (J,K) Luciferase assays using the mouse Shp promoter (from −492 to +36) showing that Shp is a direct transcriptional target of XBP1s. (J) Sequence of mouse Shp promoter with a mutated (mut) or deleted (del) putative XBP1s-binding site. (K) Cotransfection assay in HEK293T cells expressing luciferase reporter plasmids driven by Shp promoters as indicated and an XBP1s-expressing plasmid. (L) Chromatin immunoprecipitation (ChIP) and qPCR analysis showing enrichment of XBP1s at the Shp and BiP promoters. 266-6 pancreatic acinar cells were transfected with an XBP1s-Flag-expressing plasmid or empty plasmid (control [CON]). ChIP was performed with normal rabbit IgG (IgG) or anti-Flag antibody. The positions of qPCR amplicons relative to the transcription initiation site of each locus are indicated. Representative data of two experiments are shown. In B–K, data are represented as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 by unpaired two-tailed Student's t-test for two-group comparison or by two-way ANOVA analysis for multigroup comparison. See also Supplemental Figure S1.