VSG silencing without ES-attenuation is not sufficient to trigger stumpy differentiation.

<p>The experiments were conducted with a proliferating clone of the GFP:PAD1_UTR reporter cell line at densities below 5x 10⁵ cells/ml (except the density-induced control). (A) The cell cycle position of DAPI-stained trypanosomes was analyzed after 24 and 48 hours of tetracycline induction. Non-induced slender (0 h) and density-induced stumpy cells (st) served as controls. Values are given as percentages (± SD) of two experiments (total n > 700). (B) On the left DAPI staining (grey) illustrates different dividing stages (indicated by yellow arrowheads). The green GFP:PAD1_UTR stumpy marker signal is absent. The DIC image on the right illustrates the typical slender morphology of proliferating ectopic VSG overexpressors. Note the characteristic extended free part of the flagellum (white arrowhead). Scale bar: 10 μm. (C) Western blot stained with an antibody against the mitochondrial lipoamide dehydrogenase (LipDH, green), whose expression increases during stumpy development. This reveals the uniformly low LipDH expression in proliferating ectopic VSG overexpressors during the time course of induction. Detection of paraflagellar rod (PFR) proteins served as a loading control (magenta).