Fig. 3: Helicobacter pylori and CagA downregulate DLC1 expression in vitro.

A Partial colocalisation of DLC1 with MIST1+ stem/chief cells. Detection of DLC1 protein in murine gastric chief/stem cells. FFPE sections from stomachs of mock (control) and WT PMSS1-infected (6 months) mice (from Fig. 2C) were co-stained using DLC1 and MIST1 Abs for immunofluorescence microscopy. Quantitative analyses (left) and representative images (right) of the corpus region. Data are numbers of double-positive cells per mm² presented as mean (n = 2–9 uninfected control mice per time point, n.c. = no double pos. cases detectable in infected mice). Colour code: Green = DLC1; Red = MIST1; Blue = nuclei (DAPI); original magnifications ×200; Scale bar = 20 and 100 µm. White arrow/frame: Yellow = zoomed-in overlay (DLC1+/MIST1+). Insert (top left): Representative images of PCR-amplification products and murine DLC1 protein isoforms (GC: 70 kDa vs. NT: 170 kDa) from total tissue lysates detected by Western blot using Abs against the N-terminal part of DLC1 including the SAM domain (source file: “Original Western Blots” page 4). Legend: STO = Stomach (Corpus); L = Liver (pos. control); GC = gastric cancer. B Colocalisation of DLC1 with ECL cells, but not with parietal cells. FFPE sections from stomachs of uninfected mice were co-stained using DLC1, chromogranin A (CHGA), and H⁺K⁺ATPase (HK) Abs for immunofluorescence microscopy. Quantitative analyses from images of the corpus region presented in S3. Data are numbers of single or double-positive cells per field presented as mean (*p < 0.05, Kruskal–Wallis test, n = 2–9 uninfected control mice, n ≥ 5 fields per image). C Scheme of promoters identified in the human DLC1 gene according to Low et al. [7]. Note that the promoters generate four different transcripts/protein isoforms with identical C-terminus but distinct N-termini. Legend: E = Exon. D H. pylori downregulates DLC1 mRNA. AGS cells were infected with CagA+ injection-proficient H. pylori strain G27 (MOI = 100) for 3 days. Ct-values from RT-qPCRs on total RNA were normalised to B2M and calculated as % ± S.E. (*p < 0.05 vs. mock, 2way-ANOVA with Bonferroni post-tests, n = 3). E CagA is sufficient for DLC1 promoter inhibition. Cells (HEK293T, AGS, N87) were co-transfected for 72 h with EV or CagA expression plasmid and reporter vector pGL3 containing the human proximal promoter sequences of DLC1v1 or v1. Luciferase activity was normalised to protein concentration and calculated as -fold ± S.E. (*p < 0.05 vs. EV, one-sample t test, n = 3 per cell line).