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. 2017 Jan 1;130(1):219–231. doi: 10.1242/jcs.184291

Fig. 4.

Fig. 4.

Pax8 depletion disrupts apical membrane orientation, lumen formation, and basement membrane and polarized cytoskeleton organization in FRT 3D structures. (A) Protein expression levels of Pax8 in FRT cells after infection with lentiviruses containing a non-silencing shRNA (shCtr) or shRNA against Pax8 (shPax8) expression vector. Protein levels were quantified as percentages relative to shCtr expression levels (shown under blots). (B) Apical–basal polarization in shCtr and shPax8 FRT cells that had been grown for 2 and 5 days in 3D Matrigel culture. Representative single confocal sections through the middle of FRT structures that had been stained for polarity markers ezrin (green), β-catenin (red) and ZO-1 (magenta, white arrows indicate basal ZO-1 localization) are shown. (C) Quantification of lumen formation and ezrin localization. Graph denotes the percentage of shCtr or shPax8 FRT structures that formed a central lumen and exhibited ezrin delocalization at the periphery at 5 days of growth. Values are mean±s.d. from three replicates, n≥30 follicles per replicate. **P<0.001 (t-test). (D) Localization of endogenous laminin-332 in control and Pax8-depleted structures. Representative single confocal sections in the middle of shCtr and shPax8 FRT structures at 2 and 5 days of growth that had been stained for ezrin (green) and laminin-332 (Laminin, magenta) are shown. (E) Relative mRNA expression levels of β1, β2, β3, γ1 laminins in shCtr and shPax8 FRT cells. Quantitative data was related to expression in shCtr cultures (relative units). s.d. between triplicates is depicted with positive error bars. (F) Protein expression of β1-integrin in Pax8-depleted structures. Lysates were immunoblotted for Pax8 and β1-integrin (Itgb1). Protein levels were quantified as percentages relative to shCtr expression levels (shown under blots). All lysates were immunoblotted for β-actin to ensure equal loading. (G) Polarized organization of actin and microtubule cytoskeleton in control and Pax8-depleted structures at the indicated days of growth. Representative single confocal sections in the middle of shRNA FRT structures that had been stained for phalloidin (magenta) and acetylated α-tubulin (Ac-tub; green). All nuclei were stained with DAPI (blue). Scale bars: 10 µm.