Figure 2: H. pylori CagA induces EMT through stabilization of Snail protein.

(a) The CagA, endogenous Snail and phospho-Tyr (pTyr) levels were detected after treatment of 60190 or isogenic CagA deletion mutant strain (ΔCagA) in AGS and MKN28 cells (left panels). Relative protein abundance below the blot. Blots are representative of two independent experiments. The time-dependent abundances of Snail were determined by immunoblot analysis after infection with 60190 at the indicated time periods (right panels). Tubulin served as loading control. (b) E-cadherin proximal promoter activity (left panel) and E-cadherin transcript levels (right panel) were determined in 60190 or isogenic CagA deletion mutant strain in AGS cells. Activity of the E-cadherin promoter construct was normalized to the activity of the co-transfected SV-40 renilla activity. The relative abundance of E-cadherin transcripts in 60190 or CagA-deleted 60190 treated cells was compared with GAPDH. Relative reporter activity and mRNA abundance were determined from triplicate experiments (**P<0.01 by Student’s t-test). Error bars indicate s.d. (c) Following the control or 60190 infections, Snail protein half-life was determined in AGS cells at the indicated time periods after treating the cycloheximide as described in Methods. The half-life of Snail was determined from the slope of densitometric protein abundance (right panel). (d) The Snail was stably knocked down with lentivirus in MKN28 cells (dsRed-shSnail), and the E-cadherin expression levels were detected by immunoblot analysis followed by CagA-positive 60190 infections or isogenic deletion strains for 24 h. Blots are representative of two independent experiments. (e) Confluent AGS cells of control dsRed and shRNA-mediated Snail knock-down cells were wounded using a pipette tip and then infected with control or 60190 strain for 4 h (left panels). Distance between cell borders was measured from five independent sites and the relative migratory inhibition by Snail knock-down compared with dsRed control cells was analysed using two-way analysis of variance test (right panel). Error bars indicate s.d.