Fig 2: DLC1 rescues human GC cells from CagA-related adhesion defects.(A) AGS/EV cells were transiently transfected with pT-DLC1v4 plasmid for 24 h, followed by infection with CagA-delivery competent H. pylori G27 for additional 16 h and staining for immunofluorescence microscopy; green = actin (phalloidin), blue = nuclei. Magnification ×630. (B–C) Representative immunofluorescence images (A) are presented together with quantitative analyses of “humming bird” (B) and cell spreading (C) morphologies. Cell phenotypes were counted for at least 10 cells per field (total of 10 fields) and calculated as % ± S.E. (n = 3 experiments) of total cells; p = 0.0067 AGS/EV versus AGS/DLC1.

Fig 3: NEDD9 as DLC1 interacting protein is required for the polarized expression of DLC1. a Venn diagram showing the number of proteins enriched by DLC1. b DLC1-associated proteins involved in major canonical pathways as assessed by the Ingenuity Pathway Knowledge base. c Interactome network of DLC1 and its associated proteins involved in cell motility and cytoskeleton regulation. d Immunoprecipitation (IP) was performed in neural tube lysate using anti-DLC1, and IgG as control. Immunoprecipitated proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and Western blotting was performed with anti-NEDD9 and anti-DLC1 antibodies. e Schematic showing the design of the C-terminally truncated DN-DLC1 constructs. IP with anti-V5 was performed in lysates extracted from neural tubes electroporated with the indicated constructs followed by western blot with anti-V5 or anti-NEDD9. White arrowhead indicates the specific protein band of DN-DLC1 (SAM). f In situ hybridization for NEDD9, DLC1, SOX9 and SOX10 on transverse sections of st 11 chick embryo. Immunofluorescence for NEDD9, SOX9 and SOX10 on transverse sections of st 11 chick embryo. Scale bar: 50 µm. g Immunofluorescence for NEDD9 and phalloidin on emigrating NCCs from neural tube explants and nuclei are stained with DAPI. Polarized expression of NEDD9 is shown in pseudocolour. Line scan analysis showing the average fluorescence intensity of NEDD9 along the white dotted line (n = 103/11 explants). h Immunofluorescence for DLC1 and phalloidin on NCCs expressing control morpholino (Ctrl-MO) (n = 65/11 explants) or NEDD9-MO (n = 112/12 explants). Distribution of DLC1 expression in both treatments are shown in pseudocolour. Scale bars, 20 µm. i Line scan analysis showing the average fluorescence intensity of DLC1 in NEDD9-MO-treated NCCs (n = 112/12 explants)

Fig 4: Knockdown of DLC1 reverses the effects of miR-301a downregulation in NSCLC cells. (A) Expression levels of DLC1 in H1299 and A549 cells transfected with DLC1 siRNA or NC siRNA were analyzed by western blotting. Knockdown of DLC1 markedly reversed the carcinogenic effects of miR-301a on (B) proliferation, (C) migration and (D) invasion of NSCLC cells. Magnification, ×200. *P<0.05 vs. the NC inhibitor. miRNA, microRNA; NC, negative control; NSCLC, non-small cell lung cancer; siRNA, small-interfering RNA.

Fig 5: SOXE factors regulate DLC1 and NEDD9 expression. a In situ hybridization for DLC1 on transverse sections of embryos electroporated with SOX10 at 12 hpt (n = 9). Scale bar, 50µm. b In situ hybridization for DLC1 on embryos electroporated with Ctrl-MO (n = 10) or SOX10-MO (n = 10) at 24hpt. Black arrows indicate loss of DLC1 expression on the transfected side of the neural tube. Scale bar, 10µm. c In situ hybridization for FOXD3 on embryos electroporated with SOX9 (n = 9) or SOX9 + DN-DLC1 (n = 9) at 24hpt. Scale bar, 20 µm d In situ hybridization for FOXD3 on embryos electroporated with SOX10 (n = 10) or SOX10 + DN-DLC1 (n = 10) at 24hpt. Scale bar, 10 µm. e Immunofluorescence for NEDD9 and in situ hybridization for NEDD9 on transverse sections of embryos electroporated with SOX9 (n = 12) at 6 hpt. Scale bar, 50 µm. f Immunofluorescence for SOX9 and in situ hybridization for NEDD9 on transverse sections of embryos electroporated with Ctrl-MO (n = 9) or SOX9-MO (n = 9). White arrowhead indicates loss of SOX9 expression on the transfected side. Scale bar: 50 µm. g Schematic diagram showing the seven exons (black) of chick NEDD9 gene with promoter region in blue, -9.5 kb enhancer 1 (E1 harboring two putative SOX9 binding motifs, B1 and B2, and + 45 kb enhancer 2 (E2) containing a putative SOX9 binding motif, B3. h Fold enrichments of three independent ChIP-qPCR assays for SOX9, SOX10 and IgG (control) antibodies on B1, B2 and B3. Data represented as fold enrichments for putative SOX9 binding motifs relative to control. Mean±s.e.m. Bonferroni multiple comparison test, *p < 0.05, **p < 0.001, ***p < 0.0001. i Fold activation of three independent in vivo luciferase assays for reporter constructs carrying NEDD9-promoter, E1 or E2. Mean±s.e.m. Bonferroni multiple comparison test, **p < 0.001, ***p < 0.0001. j Immunofluorescence for HNK-1, Laminin and in situ hybridization for SOX103’UTR on transverse sections of embryos electroporated with SOX9 or SOX9 + NEDD9-MO at 24hpt. White arrowhead indicates loss of Laminin expression on the basement membrane of the transfected neural tube. Scale bar: 50 µm. k Quantification of the number of emigrating GFP+ NCCs expressing HNK-1 in embryos electroporated with the indicated constructs. Mean±s.e.m. Bonferroni multiple comparison test, *p < 0.05; **p < 0.001; ns, not significant