Evidence for the involvement of NOD2 in regulating colonic epithelial cell growth and survival

Figure 5 Neutralization of CARD15 gene expression in colon carcinoma cells reduces their survival. A: The level of NOD2 mRNA in HT-29 cells transfected with green fluorescent (GFP) containing lentiviral vectors expressing either NOD2 (NOD2 shRNA) or scrambled NOD2 shRNA sequences (Ctrl. shRNA) was determined by real time RT-PCR after 4 h stimulation with TNFα. Levels were compared to that of nontreated cells (= 1.0). Data are averaged values (± SE) of 3 experiments. B: The impact of CARD15 and control shRNA on the growth of HT-29 cells was assessed by determining the frequency of GFP⁺ and GFP-cells by flow cytometry at 1 d, 3 d and 6 d post infection. Data are averaged values (± SE) of 3 experiments. ^cP < 0.01. C: Frequency of apoptotic cells in cultures of non-infected HT-29 cells (Media) and in cells 2 d after infection with NOD2 shRNA or Ctrl shRNA was determined by staining with Annexin V and 7AAD and flow cytometry. The graph represents the averaged frequency (± SE) of apoptotic cells from three experiments. ^aP < 0.05. D: The survival of SW480 cells after transfection with lentiviral vectors expressing either NOD2 or scrambled shRNA sequences was analysed by determining the frequency of GFP⁺ cells remaining at various times after infection by flow cytometry. The data is representative of two independent experiments.