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Fig. 1 | Genome Biology

Fig. 1

From: Gut bacterial metabolites modulate endoplasmic reticulum stress

Fig. 1

XBP1s-GFP screen identifies microbiome molecules with ER stress-modulating activities. a The XBP1 knock-in reporter construct in HT-29 cells encodes the 261 amino-acid (a.a.) XBP1u protein in homeostatic conditions. ER stress induces splicing of a 26-nucleotide fragment (red region) within XBP1 mRNA and leads to the production of 376 a.a. XBP1s protein and self-cleavable GFP. b Green fluorescence in HT-29 XBP1s-GFP reporter cells treated with DMSO or tunicamycin (Tm) was visualized by high content imaging. Relative fluorescence of XBP1s-GFP reporter cells in the presence of the specified Tm concentrations were normalized to DMSO-treated cells (0.5% v/v). c A library of chemical compounds (the “microbiome box”) relevant to microbiome and IBD were curated and screened for ER stress activation or dampened UPR response in the presence of Tm. d The screen identified eight compounds that induce fluorescence and three others that inhibit XBP1s-GFP. Validation by HSPA5 and DDIT3 mRNA expression confirmed activity of three molecules (A5, A7, and B3). e Five molecules specifically increased fluorescence in XBP1s-GFP reporter cells when supplemented at 50 μM, indicated by ratio of fluorescence in the reporter lines to parental HT-29 (XBP1s-GFP/WT). f Fluorescence induction by 0.75 μg/mL Tm (relative to no Tm treatment) in XBP1s-GFP reporter cells was measured when supplemented by DMSO, Myc II (positive control) or 50 μM of UPR inhibitors. In all bar graphs, error bars represent standard errors of the mean from three experimental replicates, and one-way ANOVA was used for statistical analyses (asterisks represent P < 0.05)

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