WT controls for all panels are presented in Fig. In Hrs, Stam l(2)gl triple mutant cells Hnt expression and failure to downregulate Cut expression is visible only in multilayering cells that are likely not reached by the ligand, indicating that Notch activation is not affected in mutant cells that are exposed to the ligand and Notch is not ectopically activated in those that are not. Hrs Stam l(2)gl homozygous cells are marked by the absence of GFP. (N–O) Mosaic egg chambers at stages 5–7 of oogenesis stained for Hnt and Cut. Notch receptor fails to be degraded in mutant cells and it remains accumulated intracellularly after 5 hrs after the endo of labeling (0 hrs). (L–M) Endocytic trafficking assay with anti-Notch ECD to label Notch at the surface of living Hrs Stam l(2)gl mutant eye disc. Notch ECD is mainly accumulated in early endosomes in GFP-negative Hrs, Stam l(2)gl triple mutant tissue, but not in (2)gl mutant tissue. (I–K) Co-localization with anti Notch ECD (NECD) or Notch ICD (NICD) and Avl, marking early endosomes, in mosaic eye imaginal discs. Cells homozygous for Hrs Stam l(2)gl show accumulation of ubiquitin, Notch and Domeless intracellularly. (E–H) Hrs Stam l(2)gl mosaic eye and FE cells (marked by the absence of GFP) stained to detect ubiquitin, Notch and the Domeless receptors.
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(D) Eye imaginal disc formed by mutant cells homozygous for Hrs, Stam l(2)gl stained to detect sub-cortical f-Actin show a tumor-like phenotype. In Hrs Stam l(2)gl mutant tissue (GFP negative) apoptosis is activated. (C) Mosaic eye imaginal discs stained with antibody anti-activated Caspase 3 to mark apoptotic cells. Eye imaginal cells homozygous for the mutations also show mesenchymal-like cells and autonomous disruption of epithelial organization. FE cells homozygous for Hrs, Stam l(2)gl (marked by the absence of GFP) in a stage 5–6 egg chamber are misshapen and multilayered. (A–B) Epithelial disorganization in Hrs, Stam l(2)gl triple mutant tissues revealed by staining to detect sub-cortical f-Actin. Associated Data Supplementary Materials Figure S1: l(2)gl is responsible for the loss of tumor suppression phenotype in triple mutants. This article has been cited by other articles in PMC.