Figure 2.
NAG-1/GDF15 is highly induced in HCT116 cells by sulindac sulfide and DM-sulindac sulfide. A, sulindac sulfide, DM-sulindac sulfide (left), and DM-sulindac (right) at indicated doses induced NAG-1/GDF15 mRNA expression in HCT116 cells in a dose-dependent manner. HCT116 cells were seeded at 2 × 105 cells per well in 6-well plates. At 80% to 90% confluence, cells were treated with vehicle control (0 μmol/L), sulindac sulfide (5, 10, 15, and 20 μmol/L), DM-sulindac sulfide (5, 10, 15, and 20 μmol/L), or DM-sulindac (50, 100, 150, and 200 μmol/L) for 24 hours in serum-free McCoy’s 5 medium. Cells were collected and subjected to real-time PCR analysis. Data were obtained from 3 independent experiments. B, Western blot analysis showing that sulindac sulfide or DM-sulindac sulfide at 5, 10, 15, and 20 μmol/L of treatment for 24 hours induced NAG-1/GDF15 upregulation compared with vehicle control in HCT116 cells. Cell lysates from HCT116 cells transiently transfected with NAG-1/GDF15 plasmid was used as positive control. β-Actin was used for loading control. C, sulindac sulfide and DM-sulindac sulfide treatment at 20 μmol/L induced NAG-1/GDF15 mRNA upregulation in a time-dependent manner. HCT116 cells at 80% confluence were treated with 20 μmol/L of sulindac sulfide or DM-sulindac sulfide for 0, 3, 6, 9, 12, 18, 24, 36, and 48 hours. Cells were collected and subjected to real-time PCR. HCT116 cells treated with vehicle control were also collected at the above indicated time points when collecting treated cells. The results represent mean ± SD.