Fig. 1.

NID-1 induces cell death in diverse cell types. (A) Schematic of flow diagram used for identification of NID-1; structure of NID-1 (right panel). (B) BJeLR cells were treated with NID-1 (10 μg/ml) alone or co-treated with the protein synthesis inhibitor cycloheximide (CHX, 3 μM); cell viability was measured by a trypan blue dye-exclusion assay 8 h after treatment. Data represent mean ± S.D. of an experiment performed in duplicate, * (p< 0.05, left panel). The dose-response for cell death induced by NID-1 in BJeLR and HT-1080 cells was determined using the Alamar blue viability assay (right panel). (C) Time course of NID-1 (10 μg/ml)-induced cell death in BJeLR cells (left panel). Phase contrast micrographs of untreated BJeLR cells (middle) and after 8 h NID-1 (10 μg/ml) treatment (right panel). (D) BJeLR cells were treated with NID-1 and the supernatant (unattached fraction) was removed, and the cell viability for the attached fraction was determined by trypan blue exclusion. U2OS cells do not show cell detachment upon NID-1 treatment, and cell viability in these cells was assayed 30 h after NID-1 treatment, * (p< 0.05). (E) BJeLR or HT-1080 cells were seeded in 6-well plates, allowed to adhere overnight, and treated with NID-1 (5 μg/ml) for time points indicated. Cells were then trypsinized and re-plated into 0.3% agar-media mixture. Cells were stained with 0.005% crystal violet after culture for two weeks and the number of colonies per well was counted (left panel) and imaged (right panel), * (p<0.05). Data is representative of two independent experiments. (F) Time course of cell viability upon NID-1 treatment (10 μg/ml) in the indicated cell lines using trypan blue dye-exclusion assay. The experiments are representative of at least two independent experiments.