Fig. 6. During encystation, ADI is translocated to the nuclei and inhibits CWP expression. (A) Results of western blotting that was performed after cytoplasm- and nuclear-fractionation assays using anti-ADI pAb at 24 and 48 hours post-encystation induction in wild-type (ADI) cells. L, cell lysate previous fractionation; C, cytoplasmic fraction; N, nuclear fraction (upper panel). Middle panel: anti-VSP1267 mAb is used to detect cytoplasm- or nuclear-fraction contamination. Lower panel: a representative time course of IFA shows ADI (red) distribution during encystation (ESVs in green). (B) Confocal microscopy and IFA using wild-type cells show that ADI (red) is translocated to the nuclei when the encysting cell is filled with ESVs (arrowheads). CWP1 is stained in green. (C) Top panels: representative differential interference contrast (DIC) and DAPI-staining images show a stable ADI-transgenic culture (Merge). Middle panels: direct IFA shows that trophozoites highly overexpressing ADI-HA (green) in the nuclei do not express CWP2 (red) during encystation (arrowheads in Merge). Bottom panels: closer analysis of three ADI-transgenic cells demonstrated that those expressing ADI-HA (green) in the nuclei (arrowheads in Merge) do not reveal CWP2 (red) expression after 24 hours of encystation, whereas those with low ADI-HA expression do (arrow in Merge). Scale bars: 10 µm.