Journal: bioRxiv
Article Title: Epithelial sensing of vitamin A shapes intestinal antimicrobial defense
doi: 10.64898/2026.03.08.710399
Figure Lengend Snippet: (A) Vitamin A-derived retinol is metabolized to RA, which activates retinoic acid receptors (RARs). RARs bind target gene promoters to drive RA-dependent transcription. (B) HT-29 cells (human intestinal epithelial cells) were treated with the RAR antagonist BMS493 or the RAR agonist Ch55 and simultaneously stimulated overnight with RA and IL-22. REG3G transcripts were quantified by qPCR. Each data point represents an independent experimental replicate (n=6 per group). (C) HCT-116, a transfection-competent human intestinal epithelial cell line expressing RARA and RARG , was treated for 24 hours with an siRNA targeting either gene and then stimulated overnight with retinol and IL-22. REG3G transcripts were quantified by qPCR. Each data point represents an independent experimental replicate (n=4 per group). (D) IEC-specific disruption of RAR signaling using a dominant-negative RAR (dnRAR) knock-in allele. dnRAR mice harbor a loxP -flanked STOP cassette upstream of a dominant-negative RAR open reading frame. The dnRAR is derived from a mutant human RARα (RAR403) lacking the ligand-dependent transactivation domain and functions as a pan-RAR inhibitor. Crossing dnRAR mice with Villin-Cre transgenic mice excises the STOP cassette in IECs, resulting in IEC-selective expression of dnRAR and inhibition of RAR signaling. (E) qPCR analysis of Reg3g expression in small intestines of conventional dnRAR fl/fl (n=12) and dnRAR IEC (n=17) mice from five litters, and germ-free wild-type mice (n=21). (F) Immunofluorescence microscopy of REG3G in small intestines of dnRAR fl/fl and dnRAR IEC mice. Sections were stained for REG3G and counterstained with DAPI. Scale bar, 100 μ m. Images are representative of at least three fields per sample and two independent experiments (three littermates per group). (G) Mean fluorescence intensities of at least 150 villi from the images represented in (F) were quantified across at least two mice of each genotype. RAR, retinoic acid receptor; RA, retinoic acid; IEC, intestinal epithelial cell; REG3G, regenerating islet-derived protein 3γ; siRNA, small interfering RNA; dnRAR, dominant negative retinoic acid receptor; Conv, conventional; GF, germ-free. Means ± SEM are plotted; *p < 0.05; **p < 0.01; ***p<0.001; ns, not significant by Mann-Whitney test. See also .
Article Snippet: Pharmacologic modulation of retinoic acid receptor (RAR) activity was performed using the pan-RAR antagonist BMS493 (Fisher Scientific) or the pan-RAR agonist Ch55 (Tocris).
Techniques: Derivative Assay, Transfection, Expressing, Disruption, Dominant Negative Mutation, Knock-In, Mutagenesis, Transgenic Assay, Inhibition, Immunofluorescence, Microscopy, Staining, Fluorescence, Small Interfering RNA, MANN-WHITNEY
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