Supplementary Figure 1. The CagA-dependent wound healing or transwell migration of gastric cancer cell. AGS cells transfected with vector control or 3xflag-CagA expression vector were wounded using a pipette tip, and then examined cell migration for 8 h (upper left panels, scale bars 500 μm). The distances between cell borders were measured from 5 independent sites (upper right panel); the results are expressed as the mean ± s.d. (**p<0.01 by Student s t-test). AGS cell migration across transwell membrane after infection with 60190 (middle panels) or transfected with CagA expression vector (lower panels) relative to cells of negative control was determined after 24 h. Quantitative analysis of the migratory cells were counted from 5 independent sites (right panels, **p<0.01 by Student s t-test).
Supplementary Figure 2. CagA-dependent EMT in gastric epithelial cells. The AGS cells were transfected with CagA expression vectors, and the endogenous Snail protein abundance and E-cadherin proximal reporter activities were detected by immunoblot analysis (a) and reporter assay of triplicate experiment (b), respectively (**p<0.01 by Student s t-test). (c) The relative mrna expression of epithelial markers (occludin and claudin) and mesenchymal markers (vimentin and fibronectin) in 60190 infected or CagA-deleted strain (ΔCagA) infected were compared to that in control cells. Relative abundance was determined from triplicate experiment (**p<0.01 compared to control and ΔCagA, *p<0.05 compared only to control by Student s t-test). (d) Immunoblot analysis of E-cadherin repressors in AGS and MKN28 cells following treatment of 60190 or the isogenic mutant strain. Tubulin is shown as the loading control. (e) 293 cells were transiently transfected with HA-tagged Slug vectors in absence (-) or presence (+) of Flag-tagged CagA expression vector and the protein abundance of Slug was detected by immunoblot analysis. (f) The AGS and MKN28 cells were stably transfected shrna against control (dsred-control) or Snail (dsred-shsnail) with lentiviral transduction as described in the Methods section. The endogenous expression levels of Snail were detected by immunoblot analysis.
Wound healing assay (x40) 0h 4h 6h 60190 60190 AGS dsred-shsnail control AGS dsred control Supplementary Figure 3. Effect of Snail knock down on AGS cell migration. Confluent AGS cells of dsred control or shrna-mediated Snail knock-down were wounded using a pipette tip and then infected with an H. pylori 60190 for 4 h or 6 h. scale bars; 100 μm.
Supplementary Figure 4. Binding of CagA and GSK-3. (a) The gastric cancer cells were infected with control or 60190 for 8 h, and GSK-3α/β and pser9-gsk-3β protein abundance in soluble and insoluble fraction were detected by immunoblot analysis. (b) The 293 cells were transfected with 3xflag-CagA expression vectors for 48 h. The lysates were subjected to immunoprecipitation with anti-flag agarous, and then binding ability of GSK-3 and pser9-gsk-3β on CagA was detected with immunoblot analysis. CagA-PR denotes tyrosine phosphorylation resistant mutant on EPIYA motifs. (c) The AGS cells were transfected with HA-tagged GSK- 3β and flag-tagged CagA expression vectors for 48 h. The cells were stained with anti-flag (green) and anti-ha (red) for visualization by confocal microscopy (upper panels). The scale bar represents 10 μm. The insets were enlarged to observe colocalization of CagA and GSK-3 (lower panels); white arrows indicate co-localized membranous foci of GSK-3 and CagA.
Supplementary Figure 5. (a) The independent localization of CagA and Rab9, a marker of late multivesicular endosome. Following transduction of CagA into the AGS cells, intracellular localization of CagA and endogenous Rab9 was detected by immunofluorescence as described in Fig. 4. The scale bar represents 10 μm. (b) Full-length CagA was co-transfected with HA-tagged wt GSK-3β or mutant of V267G/E268R in 293 cells. The soluble cell lysates and insoluble fractions were subjected to immunoblot analysis.
Supplementary Figure 6. Routine H/E section and Snail protein expression detected by immunohistochemistry (IHC) in H. pylori (-) or (+) gastritis samples included in Fig. 7. The scale bar represents 10 μm.
Supplementary Figure 7. Full scans of uncropped SDS-PAGE blot. Boxes denote lanes used in Figures
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Supplementary Table 1 Primer oligonucleotide sequnences for real time RT-PCR analysis Genes Forward Reverse E-cadherin 5 TGAGTGTCCCCCGGTATCTTC 5 CAGTATCAGCCGCTTTCAGATTTT Occludin 5 CGGTCTAGGACGCAGCAG 5 AAGAGGCCTGGATGACATGG Claudin 5 GGCTGCTTTGCTGCAACTGTC 5 GAGCCGTGGCACCTTACACG Vimentin 5 TTTTTCCAGCAAGTATCCAACC 5 GGAGTTTTCCAAAGATTTATTGAA Fibronectin 5 CAGGATCACTTACGGAGAAACAG 5 GCCAGTGACAGCATACACAGTG GAPDH 5 - ATGGGTGTGAACCATGAGAAG 5 - AGTTGTCATGGATGACCTTGG