Fig. 5

KLF4 was bound to the promoter of NUCB2 in melanoma cells. a Schematic illustration of pGL3-based reported constructs were used in luciferase assays to examine the transcriptional activity of NUCB2. b The promoters of NUCB2 named P1, P2 and P3 were individually transfected into Mel-RM cells with or without TM treatment. The luciferase activity was measured. The data represent the means ± SD of three independent experiments; ***p < 0.001 vs. control. c P3 was transfected into Mel-RM cells with or without KLF4 knockout. The cells were treated with 3 μM TM. The luciferase activity of NUCB2 promoter was measured by the luciferase reporter assay. The data represent the means ± SD of three independent experiments; ***p < 0.001 vs. control. d-e P3 was transfected into Mel-RM and A375 cells with or without KLF4 knockdown. The cells were treated with 3 μM TM. The luciferase activity of NUCB2 promoter was measured by the luciferase reporter assay. The data represent the means ± SD of three independent experiments; **p < 0.01 vs. control. f The potential KLF4-binding sites were inspected by JASPAR. A schematic illustration of KLF4 wild type binding site 1 (BS1), binding site 2 (BS2) and the matching mutant BS1M, BS2M that were used in the luciferase assays is shown. g BS1, BS1M, BS2 and BS2M were individually transfected into Mel-RM cells with or without 3 μM TM treatment. The luciferase activity was measured by the luciferase reporter assay. The data represent the means ± SD of three independent experiments; ***p < 0.001 vs. control. h BS1 was transfected into Mel-RM with or without KLF4 knockout. The cells were treatment with 3 μM TM. The luciferase activity was measured by the luciferase reporter assay. The data represent the means ± SD of three independent experiments; ***p < 0.001 vs. control. i-j ChIP analysis showed the binding of KLF4 to the promoter of NUCB2 in KLF4 WT or KO Mel-RM cells in response to 3 μM TM treatment. An isotype-matched IgG was used as a negative control