Impairment of cholesterol uptake in <i>cup-1</i> mutant animals and expression of <i>cup-1</i> in <i>C. elegans</i>.

<p>(A) Animals were grown in the presence of the cholesterol analog NBD and fluorescence was assessed by confocal microscopy. The upper panel shows a representative N2 wild-type animal. DAPI signal is shown in blue. Rectangles indicate zoom areas to the tail, mid section and head (only green channel). The lower panel shows a representative <i>cup-1(gk245)</i> animal. Scale bar: 100 µm. (B) Boxes represent first to third quartiles around the median of green channel intensity of wild-type animals (WT) fed with an empty plasmid in comparison to <i>cup-1(gk245)</i> and RNAi animals grown in the presence of NBD. Mock: basal autofluorescence from animals grown without NBD. The mean basal autoflurescence value of mock animals was taken as zero. Different letters [a], [b] and [c] indicate statistically significant differences per parameter among experimental groups, p<0.05. Bars: min. and max values; A.U: arbitrary units; n>20 for each condition. (C) The BC12913 strain (expressing GFP under <i>cup-1</i> promoter) was grown in the presence of the fluorescent cholesterol analog DHE and colocalization was assessed by confocal microscopy. Upper panels show Nomarski image overlapped with the green channel where GFP signal is observed in the pharynx, nerve ring and intestine cells (arrows). DHE signal is shown in blue. The rectangle indicates the zoom area of intestine cells presented in the lower left panel. A colocalization channel (lower right panel) was built from positive PDM values resulting from both pixels above the mean. <i>t</i>: tail; <i>h</i>: head; phx: pharynx; piv: pharyngeal-intestinal valve. Scale bar: 50 µm. (D) RT-PCR of <i>cup-1</i> transcript evaluated during larval development. GADPH (<i>gpd-3</i>) was used as control. No difference in <i>cup-1</i> expression was observed over time. Numbers indicate the rate cup-1/gpd-3. L1/L2 expression was taken as 1. −RT: negative control without reverse transcriptase.