Regulated Expression of an Essential Allosteric Activator of Polyamine Biosynthesis in African Trypanosomes
Figure 6
The effects of chemical inhibition of AdoMetDC on the expression of polyamine biosynthetic enzymes.
(A) Comparison of AdoMetDC genetic knockdown with AdoMetDC inhibition by MDL 73811. AdoMetDC RNAi cells were split into fresh media and induced with Tet, or treated with 25 or 75 nM MDL73811. Cells were collected over a 24–48 h time course and analyzed by Western blot. (B) Analysis of protein and mRNA levels in 427 cells treated with MDL 73811 for 24 h. Protein (solid symbols) and mRNA (open symbols) were normalized to tubulin controls and then displayed as a fold change relative to untreated cells. The gel used for quantitation is displayed in Figure 5. AdoMetDC, green triangles; prozyme, purple squares; and ODC, orange circles. (C) The effect of cycloheximide treatment on protein expression. Cycloheximide (CHX) (50 µg/ml) and/or MDL 73811 (MDL) (75 nM) were added to the culture and cells were collected for Western analysis after 3, 4.5, and 6 hrs of drug treatment. Control cells underwent identical procedures but were not treated with either CHX or MDL73811. (D) Quantitation of protein levels of AdoMetDC, prozyme, and ODC during treatment with cycloheximide. Values are shown as the ratio to tubulin controls, normalized to untreated cells at time zero. AdoMetDC, green; prozyme, purple; and ODC, orange. Errors represent the standard error of the mean (n = 2).