Fig. 4. Nuclear CTS7 causes trophoblast proliferation and differentiation defects. (A) Differentiation of sinusoidal giant cells marked by Prl3b1 (blue) is reduced in the labyrinth layer of E12.5 Sox2-Cre.tg^Cts7 placentas. The border to the spongiotrophoblast is indicated. (B) Isolectin BSI-B₄ staining (brown) on E12.5 placentas labelling glycogen cells (Glc, encircled in red). Bracket indicates the spongiotrophoblast (SpTr) layer. Broken black lines indicate the borders to the decidua (Dec) and labyrinth (Lab). (C) Anti-phosphohistone staining (H3S10-P, brown) on E9.5 trophoblast of CMV-Crextg^Cts7 matings and (D) quantification of signals. The arrow indicates a cell in metaphase of mitosis. Arrowheads indicate some nuclei with a speckled punctate staining in the tg^Cts7 sample. (E,F) Ki-67 staining (brown) of E12.5 placentas shows denser clusters and less differentiated (negative) trophoblast cells in transgenic (Sox2-Cre.tg^Cts7) placentas. Quantification in F. (G) Proportion of H3S10-P positive TS cells 2 days post-transfection with empty vector control (vec), wild-type Cts7, nuclear localization signal-mutant (NLS) and active site-mutant Cts7 (AS). Accumulation of H3S10-P staining depends on nuclear localization and proteolytic activity. Transfected cells were identified by GFP expression. ^*P<0.05. (H) Confocal sectioning of TS cells expressing Cts7 or Cts7-NLS. CTS7 protein can be found inside the nucleus (arrows) of some cells, whereas the NLS-mutant variant is clearly excluded from this compartment. Scale bars: 200 µm in A,B; 20 µm in C; 100 µm in E; 20 µm in H.