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. 2013 Nov 21;8(11):e79432. doi: 10.1371/journal.pone.0079432

Figure 4. Evidence of β-catenin and NF-κB interplay in vitro.

Figure 4

A. Measurement of Wnt2b and Wnt5a expression in YAMC (Young Adult Mouse Colon) cells in vitro. YAMC cells (5×105) were either uninfected (N) or infected with CR at 90∶1 MOI for 3 hr. Cells were washed thoroughly to remove bacteria and incubated in fresh medium containing antibiotics for indicated period of time. Total RNA was examined for the expression of Wnt2b and Wnt5a via RT-PCR. GAPDH was used as loading control. B. Effect of Wnt2b knockdown on reporter activity. YAMC cells were transiently transfected with TOPflash plasmid and with siRNA specific to Wnt2b and Wnt5a, respectively. After 24 h, cells were infected with CR at 90∶1 MOI for 3 h, washed to remove bacteria followed by measurement of reporter activity at 48 h using Renilla luciferase as internal control (★, p<0.05 vs. N; ★★, p<0.05 vs. CR; *, p<0.05 vs. CR; n = 3 independent experiments). C. Effect of Wnt2b and Wnt5a addition on reporter activity. YAMC cells, following transient transfection for 24 h with TOPflash plasmid and with siRNA specific to Wnt2b and Wnt5a, respectively. After 24 hr, cells were incubated with purified Wnt2b or Wnt5a, infected with CR at 90∶1 MOI for 3 h, washed to remove bacteria followed by measurement of reporter activity at 48 h using Renilla luciferase as internal control (★, p<0.05 vs. N; ★★, p<0.05 vs. CR; *, p<0.05 vs. CR; n = 3 independent experiments). D. Effect of Wnt2b and Wnt5a knockdown on β-catenin nuclear localization. YAMC cells were transfected with siRNA specific to Wnt2b and Wnt5a, respectively for 24 h. Cells were subsequently treated with control media or with CR (<@email>@90<@/email>∶1 MOI) for 3 hr followed by washing to remove bacteria. At 24 hr post-infection, cells were stained with antibody for β-catenin while nuclei were stained with DAPI. E. Effect of Wnt2b and Wnt5a knockdown on wound healing. YAMC cells were transfected with siRNA specific to Wnt2b and Wnt5a, respectively for 24 h. Cells were subsequently treated with control media or with CR (@90∶1 MOI) for 3 hr followed by washing to remove bacteria. At 48 h, cells were wounded with a plastic pipette tip. After removing debris and adding fresh media, cell migration was followed for 16 h. Figure 4F is a representative bar graph showing percent migration at 16 h (♦, p<0.05 vs. N; *, p<0.05 vs. CR; n = 3 independent experiments). G. Effect of Wnt2b and Wnt5a knockdown on NF-κB activity. YAMC cells were transfected with siRNA specific to Wnt2b and Wnt5a, respectively for 24 h. Cells were subsequently treated with control media or with CR (@90∶1 MOI) for 3 hr followed by washing to remove bacteria. At 48 h, cells were lysed and NF-κB activity in the nuclear extracts was examined by utilizing TransAM NF-κB p65 Chemi Transcriptional Factor assay kit from Active Motif (★, p<0.05 vs. N; ★★, p<0.05 vs. CR; n = 3 independent experiments).