50-100 micromolar cycloheximide should be heaps, it is apparently effective within 15 minutes of addition, but it would probably be best if you waited for 30 min to one hour to ensure effectiveness. As far as I recall, CHX only stops about 70-80% of protein synthesis, and emetine is more effective, though CHX is more commonly used. Ideally you will do titrations of the amount and times to use CHX before deciding on what you will do for your experiments.
AS to whether you should wash your cells after removal of CHX, yes, you don't want residue interfering with your experiment.
Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.
Selective Inhibition of FOXO1 Activator/Repressor …
AB - Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low concentration (1.5 μg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 μg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 μg/ml) induced diffuse swelling or oncosis-diffuse cell swelling (DCS). To assess the underlying mechanism of sanguinarine-induced apoptosis and oncosis-BCD in K562 cells, we studied their response to pre-treatment with two chemical compounds: aurintricarboxylic acid (ATA) and cycloheximide (CHX). The pretreatment effects of both chemical compounds on apoptosis and oncosis-BCD were evaluated by measuring multiple parameters using quantitative morphology, electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling and annexin-V-binding. ATA, a DNA endonuclease inhibitor, efficiently prevented DNA nicking and inhibited apoptosis almost completely and oncosis-BCD by about 40%, while CHX, a protein synthesis inhibitor, failed to inhibit both apoptosis and oncosis-BCD. These results demonstrate, first, the importance of endonuclease in sanguinarine-induced apoptosis and to some extent in oncosis-BCD and, second, that this inhibition does not require de novo protein synthesis.
Methods Used for Harvesting and Delaying Ripening
The inhibitors of initiation pactamycin, cycloheximide, NaF, aurintricarboxylic acid and pederine have been found to inhibit the reaction of initiator tRNA with puromycin, as measured by the formation of N-formyl[35S]methionylpuromycin. None of these inhibitors, with the exception of aurintricarboxylic acid, inhibits, however, the binding of the initiator tRNA to ribosomes, as measured by the binding of [35S]Met-tRNAf to unwashed or washed ribosomes. This binding is stimulated by the codon AUG; the stimulatory activity of this trinucleotide decreases with time even when ribosomes are kept at 0°C. The ribosomal components that bind initiator tRNA have been analyzed by sucrose gradient centrifugation; Met-tRNAf is bound by polyribosomes, 80-S ribosomes and 40-S ribosomal subunit. Those compounds that inhibit elongation as well as initiation, like cycloheximide, pactamycin and pederine, interfere presumably with a biochemical step common to both processes.
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AB - Cycloheximide is the most common protein synthesis inhibitor, and is believed to specifically inhibit the cytoplasmic protein synthesis. Here we demonstrate that cycloheximide induces internalization and redistribution of EGF receptor to early endosomes in HeLa cells independent of receptor tyrosine phosphorylation, but dependent on p38 MAPK activity. Degradation of EGF receptor or its downstream effectors was not observed. EGF-induced activation of ERK1/2 was inhibited upon pre-treatment with cycloheximide, but did not activate JNK. The observed effects of treatment with cycloheximide alone are significant and therefore results involving the use of cycloheximide for inhibition of protein synthesis must be interpreted with caution. Structured summary of protein interactions: EEA1 and EGFR colocalize by fluorescence microscopy (View interaction).