Fig. 2. Cytological characterization and effects of Cts7/Cts8 expression in TS cells. (A) Northern blot hybridization on trophoblast stem (TS) cells grown in media promoting stem cell maintenance (+FGF/CM) or differentiation (-FGF/CM). Expression of Cts7 and Cts8 correlates with the profile of giant cell markers (Cdx2, stem cells; Ascl2, ectoplacental cone and spongiotrophoblast; Prl3d1, primary, parietal giant cells; Prl3b1, secondary giant cells; Prl2c2, all giant cells). (B) In situ hybridization on TS cell grown for 4 days in differentiation medium. Some giant cells (blue, arrows) are positive. (C) Immunostaining of CTS7 showing localization to the perinuclear area, the Golgi, and to the cytoplasm in a granular pattern indicative of endo- and lysosomal localization. (D) Western blots of transfected TS cells and their supernatants. CTS7 and CTS8 are secreted into the medium; intracellular control proteins (PFPL) were not detected in the supernatant. (E,F) Relative cell size measurements of TS cells 2 days after transfection with empty GFP-expression vector and Cts7-GFP (E) or Cts8-GFP (F). Cathepsin expression causes a significant shift towards larger cell sizes. Scale bars: 40 µm in B; 20 µm in C.