Translation Inhibition in HeLa Cells

To study translation inhibition in cellula, a total of 3,000 HeLa cells (HG1-luc2-IRES-tCD cells [20 (link)]) were seeded per well in a 96-well plate (Greiner) and incubated at 37°C and 5% CO₂ for 24 h. After replacing the medium, the cells were exposed to medium supplemented with 100 nM S. nigra RIPs and SNA-II, and incubated at 37°C and 5% CO₂ for another 24 h. Cycloheximide (10 μg/ml, Sigma-Aldrich) was used as the control treatment [33 (link)]. Luciferase activity in cell extracts was assessed using the Promega Luciferase Assay System according to the manufacturer’s instructions (Promega, Mannheim, Germany). After washing with PBS, cells were lysed with lysis buffer (Promega luciferase kit) for 2 min and half of the cell lysate was transferred to the luminometer plate (Greiner). After addition of the D-Luciferin substrate, luminescence was measured for a period of 10 seconds. Recorded signals were normalized to the amount of viable cells as measured in a subsequent Presto blue assay. Four technical replicates were performed for each protein concentration, and each experiment was repeated three times.