p0071 loss phenocopies FLCN-deficiency.

<p><b>A)</b> HEK293 cells expressing control non-targeting shRNA, FLCN shRNA, or p0071 shRNA were cultured in anchorage-independent growth conditions using a rotary shaker and sheared (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047842#s4" target="_blank">Methods</a> for details). A representative phase contrast image is shown pre- and post-shear. <b>B)</b> Quantification of cell cluster size from (A) using pixel number. After shearing, 65% of the control cells sheared into clusters of less than 10,000 pixels. In contrast, only 8% (n = 3, p<0.01) of the FLCN shRNA cells and 12% (n = 2, p<0.01) of the p0071 shRNA cells sheared into clusters of less than 10,000 pixels. Data in (A) and (C) are expressed as mean ± standard deviation. <b>C)</b> Western blot analysis of HEK293 cells showing decreased levels of p0071 using two different shRNAs (79, 80). Clone 80 was used in (A). <b>D)</b> UOK257 cells and UOK257-2 cells were analyzed by immunofluorescence using pan-keratin antibodies (20×magnification). Pan-keratin immunofluorescence was stronger in FLCN-null UOK257 cells. ZO-1 was used to identify cell borders. <b>E</b>) UOK257 cells and UOK257-2 cells were analyzed by immunofluorescence using keratin-18 antibodies (60×magnification). Keratin 18 immunofluorescence was also stronger in FLCN-null UOK257 cells. <b>F)</b> pan-Keratin levels were compared by immunoblot in UOK257 and UOK257-2 cells. Keratin levels were increased in the FLCN-null UOK257 cells. <b>G</b>) Keratin-18 levels were compared by immunoblot in UOK257 and UOK257-2 cells. Keratin-18 levels were also increased in the FLCN-null UOK257 cells. <b>H)</b> Keratin levels were analyzed by immunoblot in T84 cells expressing p0071 shRNA (+) or non-targeting control (-) shRNA. Keratin levels were elevated in p0071-deficient T84 cells. <b>I)</b> FLCN and p0071 regulate lumen formation in a 3-dimensional matrigel assay. T84 cells stably expressing either non-targeting (control), FLCN, or p0071 shRNA were stained for F-actin (red) and counter stained with DAPI (blue) to visualize nuclei. Downregulation of FLCN or p0071 leads to disorganized, irregularly shaped cell clusters with smaller lumens. The bars represent 20 um.