Fig. 3

TFF1 suppresses STAT3 nuclear localization in ex vivo and 3D organoid cultures. a Ex vivo immunofluorescence staining of p-STAT3 (Y 705) in gastric epithelial cells isolated from the antropyloric region in TFF1-wild type (TFF1-WT) or TFF1-knockout (TFF1-KO) mice. As shown, there was absence of nuclear p-STAT3 staining in TFF1-WT cells (arrowheads in upper panels), whereas strong nuclear p-STAT3 was detected in TFF1-KO (arrows in lower panels). Quantification of nuclear p-STAT3 positive staining in at least 200 counted cells from three different fields is presented as percentage ± SEM (right panel); ^***P < 0.001 by two-tailed Student’s t test. b 3D organoid cultures derived from antropyloric glands of TFF1-WT (upper two panels) and TFF1-KO (lower two panels) showing nuclear localization of p-STAT3 in TFF1-KO organoids (arrows), but not in TFF1-WT (arrowheads). c Immunofluorescence assay performed on organoids derived from the antrum of TFF1-KO mouse stomach. Cells showing nuclear staining of p-STAT3 in organoids treated with conditioned media from AGS-pcDNA cell line (Ctrl CM) for 24 h (upper panel). Organoids treated with TFF1 conditioned media from AGS-TFF1 cell line (TFF1 CM) displayed loss of nuclear staining (arrowheads) of p-STAT3 (middle panel). Organoids treated with recombinant TFF1 protein (400 ng mL⁻¹) showed absence of p-STAT3 (arrowheads) nuclear staining (lower panels). The merge of the representative full organoid gland and its H&E staining is presented in the right panels. Scale bar 2 µm. Original magnification is ×600. Zo1 (red) was used as an epithelial cell marker, and DAPI (blue), as a nuclear counterstain. Graph showing the quantification of nuclear p-STAT3-positive cells in at least 4 counted organoid glands (at least 25–50 cells in each organoid) presented as percentage ± SEM (right upper panel); ^***P < 0.001 by two-tailed Student’s t test. H&E staining and bright field images (BF) of representative organoids are shown on the right of panels (b) and (c)