Fig. 6. Polarized distribution of the tight junction associated protein, ZO-1, in aggregates of Pax3^flag-expressing Saos-2 cells. The formation of apico-basal epithelial type cell polarity was assessed by examining the distribution of ZO-1 by indirect immunofluorescence confocal microscopy. The distribution of ZO-1 in control infected (a-d,m) or Ad-Pax3^flag-infected (e-l,n) cells was examined at three days postinfection. Optical sections through the regions indicated are depicted in a-c, e-h and i-l. Panels e-h are representative of the ZO-1 distribution in single layered aggregates, and (i-l) its distribution in multi-layered (this series depicts a two-layered aggregate) aggregates of Ad-Pax3^flag-infected cells. (j) Optical section through the interface between the apex of the layer of cells attached to the substratum and the basal regions of the second layer of cells. (m,n) XZ-projections of the series presented in a-d and e-f, respectively. Note that in Ad-Pax3^flag-infected cells, ZO-1 junctional staining is restricted to the apex of lateral sites of cell-cell contact (arrow in n), and in multi-layered aggregates the junctional staining is strongest at the apex of exposed cells (compare j and k). Bars, 20 µm (a,e,i). Bars, 10 µm (m,n).