Fig 1: Retinoic acid induces Vipr1 gene expression. (A) SMG organoids were treated with or without RA (300 nM) for the indicated time. After harvesting, the organoids were subjected to qRT-PCR to evaluate gene expression associated with VIP signaling (n = 4). (B) SMG organoids were treated with varying doses of RA for 7 days and subjected to qRT-PCR for the evaluation of gene expression of Vipr1 and Rarb, a positive control, for RA-mediated signaling (n = 4). (C) Expression of VIPR1 (green) in mouse SMG tissues (left) was assessed by co-staining with KRT5 (magenta, left), KRT7 (cyan, left), ACTA2 (magenta, right), and MIST1 (cyan, right). Nuclei were counterstained with DAPI (white) and each single channel image was placed below. Scale bars indicate 20 µm. (D) The mouse SMG organoids cultured with (bottom) or without RA (top) were subjected to immunofluorescence for the evaluation of VIPR1 (green), KRT5 (magenta), and KRT7 (cyan) expressions. Nuclei were counterstained with DAPI (white), and each single channel image was placed below. Scale bars indicate 50 µm. (E) The expression of several salivary gland markers in organoids treated with or without RA were analyzed using qRT-PCR at the indicated time points (n = 4). Results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. All experiments were performed at least three times independently.