Biomolecules & Therapeutics : eISSN 2005-4483 / pISSN 1976-9148

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Fig. 2. N-terminal domain consisting of 34 amino acids is necessary for NOEY2(wt)-mediated tumour growth. (A) To assess the inhibitory effect, we generated three truncated NOEY2-expressing constructs (deletion of GTP-binding domain, deletion of C-terminal and deletion of N-terminal). Ovarian tumour cells were transfected with control (empty-inserted vector only), NOEY2(wt), NOEY2 (ΔGTP BD), NOEY2 (ΔC-terminal) or NOEY2(ΔN-terminal), respectively. Transfected cells were re-plated in 100-mm diameter dishes and grown for 14 days. Numbers of crystal violet-stained colonies were counted. Data are presented as mean ± SD from three independent experiments. *p<0.05; **p<0.01 compared to control. (B) Cell distribution of early-stage and late-stage apoptotic cell death was calculated using FACScalibur. Cells were maintained on 60 mm-diameter plates and transfected with control (empty-inserted expression vector only), NOEY2(wt), NOEY2 (ΔGTP BD), NOEY2 (ΔC-terminal) or NOEY2(ΔN-terminal), respectively. Cells were treated with FITC-labelled annexin V, propidium iodide. (C) Cell proliferation was evaluated using the CellTiter-Glo assay system. SKOV-3 cells were grown at a density of 4.5×103 per well in 96-well plates. After 24 h, cells were transfected with various concentrations of NOEY2-N. Each data point represents triplicate samples, and bars indicate mean ± SD. *p<0.05 versus zero concentration. (D) Caspase-3 activity was measured using a microplate reader in fluorescence mode with 400 nm (excitation wavelength) and 505 nm (emission wavelength). Enzyme activity was evaluated and indicated as fluorescence by the formula supplied by the manufacturer. The data shown represent three independent experiments. *p<0.05 compared to control. (E) PARP cleavage activated by control, NOEY2(wt) or NOEY2-N transfection. Soluble protein extracts were prepared and visualised by western blotting for cleaved PARP. Experiments were repeated at least three times with similar results. β-actin was used as a loading control.
Biomolecules & Therapeutics 2021;29:506~518
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