FIGURE 2.

Knockdown of PHLPP alters the apical-basolateral polarity in Caco2 cells grown in three dimensions. Caco2 cells infected with shRNA lentivirus targeting PHLPP1, PHLPP2, or both PHLPP isoforms were seeded in 3D matrix as a single cell suspension and allowed to grow for 14 days. The cells were fixed and co-stained with the ZO-1 antibody (red), Alexa Fluor 488-conjugated phalloidin (green), and DAPI (blue). A, representative confocal images were taken from control and PHLPP knockdown cells showing the localization pattern of actin and ZO-1. Scale bar, 20 μm. B, the percentages of control and PHLPP knockdown cells with a single lumen were quantified based on the pattern of actin staining and expressed graphically. Fifty randomly chosen cysts were scored. The shaded bars represent cysts with one lumen, and the open bars represent cysts with multiple or filled lumens.