NUP-1 knockdown leads to increased VSG switching and represses differentiation-induced expression site repositioning.

<p>(A) Top: Cells were probed with antibodies against the active <i>VSG 2</i> (red) and an alternate <i>VSG 6</i> (green) following 96 h of NUP-1 RNAi induction. Cells switched from expressing <i>VSG 2</i> to <i>VSG 6</i> (white arrow), expressed neither <i>VSG 2</i> nor <i>VSG 6</i> (white arrowhead), or expressed both (not shown). Bar: 10 µm. Bottom: Quantitation of increased <i>VSG</i> switching in NUP-1 knockdown cells. Graph represents average and standard deviation of three independent NUP-1 RNAi lines (<i>n</i> = 1,000 cells). (B) NUP-1 depletion represses differentiation-induced repositioning of the active expression site to the nuclear periphery. Left: Representative images of maximum intensity projections of 3-D data sets of the GFP-LacI tagged active expression site (green) in wild type cells or NUP-1 RNAi cells (induced for 24 h) after induction of differentiation. Bar: 1 µm. Right: Statistical analysis of the expression site position. The active expression site rapidly repositions to the nuclear periphery in wild type cells. Repositioning is significantly inhibited in NUP-1 RNAi cells. Graph represents the average of three replicate experiments.