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FIGURE 2 NB-5-MT (2) suppresses HIF-1-mediated transactivation and ROS generation through enhanced cellular uptake. (A-B) Under hypoxia, HCT116 cells were treated with 1 mM of melatonin (M), or its derivatives 1, 2, 6, and 7 for 4 hr The expression of HIF-1α and VEGF was analyzed by RT-PCR and western blot. A, The effect of M, 1, 2, 6, and 7 on HIF-1α mRNA and protein expression under hypoxic conditions. B, The effect of M, 2, and 7 on VEGF, Glut1, and EPO mRNA expression under hypoxic conditions. β-actin was used as a loading control. C, The effect of M, 2, and 7 on VEGF protein expression under hypoxic conditions. Tubulin was used as the loading control. Experiments were performed in triplicate, and representative images are shown. D, The effect of derivative 2 on HIF-1α-mediated transactivation. HCT116, HeLa, and MDA-MB-231 cells were transfected with pSV40promoter-EpoHRE-Luc and then treated with either 1 mM of M, or 2. Values are expressed relative to vehicle-treated cells, normalized to 100. Values shown are the means ± SD and were obtained from 3 independent experiments. *P <.05 in vehicle vs. hypoxia; +P <.05, hypoxia vs. hypoxia plus M or 2 treatment. E, The effect of 2 on oxidative stress in HCT116, HeLa, and MDA-MB-231 cells. Cells were treated with either 1 mM of M, or 2 under hypoxia. Intracellular ROS were stained with a fluorescent dye (CellROX Green Reagent). Cell nuclei were counterstained with Hoechst 33 362 (blue). Experiments were performed in triplicate and representative images are shown. F, Cellular uptake of 2 in HCT116, HeLa, and MDA-MB-231 cells. Values are the means ± SD and were obtained from 3 independent experiments. *P <.05