Did B was kindly provided by the Natural Products Branch, NCI (NSC 325319, Bethesda, MD, USA), and CHX was purchased from Sigma Aldrich. Both chemicals were dissolved in DMSO for stock. To monitor translation of proteins, Jurkat cells were transfected with pCMV-Gluc 2 plasmid and seeded in 24-well plate. At 5 h post-transfection, serially diluted stocks of Did B and CHX were added at concentrations of 0.032, 0.16 and 0.8 nM. The luciferase activity in culture supernatant was measured after 8 h and 24 h of treatment using Biolux Gaussia luciferase Flex Assay kit (NEB). Cell proliferation was monitored using CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega), as instructed by the manufacturer.
Cycloheximide (chx)
Manufactured by New England Biolabs
CHX is a laboratory equipment product used for cell disruption and homogenization. It utilizes a high-speed motor to generate mechanical force for effective cell lysis and sample preparation.
Automatically generated - may contain errors
2 protocols using cycloheximide (chx)
1
Evaluating Protein Translation Inhibitors
2
Polysome Profiling of mRNA Distribution
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Polysome profiling was performed as previously described (3, 5) . Briefly, 5×10 6 HCT116 cells were cultured in a 10 cm dish for 12~16 hours. CHX (2112S, New England Biolabs) was added to the medium to a final concentration of 100 µg/mL for 10 min. Cells were collected on ice and lysed in polysome extraction buffer (20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2 and 0.5% NP-40). After centrifugation, the lysate was separated through 10% to 50% sucrose gradients, 12 fractions were collected, and RNA extracted to perform RT-qPCR. The distribution of mRNAs was quantified by RT-qPCR analysis and plotted as a percentage of the specific mRNA in each fraction relative to the total amount of that mRNA in the gradient.
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