Fig. 5.

Loss of AIM1 increases cell invasion and cell motility without significantly altering cell proliferation. a Knock-down of AIM1 in RWPE-1 cells does not alter cell proliferation. b sh-AIM1 and control RWPE-1 cells grown to a monolayer were serum starved for 12 h. After introducing a scratch, cells were cultured in growth medium for 24 h and cell migration was monitored by live cell microscopy (also see Supplementary Movies 1 and 2). Scale bars indicate 100 μm. c Quantification of cell migration experiments show robust wound closure of RWPE-1 cells depleted of AIM1 in a 24-h time frame (mean ± SD for five replicates). d RWPE-1 cells depleted of AIM1 show dramatically increased cell invasion in Boyden Chamber invasion assays. RWPE-1 sh-AIM1 and control cells were plated in transwells containing isolated extracellular matrix components (laminin and collagen), matrigel or combinations. Forty-eight hours after seeding, invaded cells were quantified (mean ± SD for four replicates). Scale bars indicate 10 μm. e This invasion phenotype can be fully rescued in sh-AIM1 RWPE-1 cells by overexpression of full-length AIM1 (WT) and the actin-binding-proficient mutant Δ1287, but not by the actin-binding-deficient mutant AIM1 Δ859 or YFP-control (mean ± SD for four replicates). f Knockdown of AIM1 increases anchorage-independent growth of RWPE-1 cells in agarose matrices (mean ± SD for three replicates). Scale bars indicate 100 μm. g Depletion of AIM1 increases invasion through a matrigel matrix of prostate epithelial cells 957 (mean ± SD for three replicates). h AIM1 depletion in 957 cells results in increased spheroid size in spheroid invasion assays. Note the increased size of spheroidal structures and the increased number in invading protrusions. Scale bar indicates 500 μm (box-and-whisker plots of five replicates, with whiskers representing the range). All P values are derived using t-test statistics. i RWPE-1 control and shAIM1 cells were grown in matrigel for 5 days. After fixation, cells were stained with AlexaFluor-labeled phalloidin (red) and DAPI (blue) and imaged by confocal microscopy. Note that RWPE-1 control cells formed defined acinar structures (white arrows) with connecting branching structures (white arrowheads). This highly organized branching/tubular architecture was disrupted in RWPE-1 shAIM1 cells, which instead showed spindly elongated protrusions (red arrows). Scale bars indicate 200 μm