Presenilin-2 localization at basal bodies is required to efficiently rescue differentiation and Notch-signaling defects in embryos and cultured keratinocytes depleted of Presenilin-1 and Presenilin-2. (A) E17.5 epidermis was transduced at E9.5 with Psen1-2 shRNA (H2B-RFP) alone or with WT GFP-Presenilin-2 or GFP-P486A Presenilin-2, and then subjected to IF microscopy with an antibody against Presenilin-2. (B and C) Images of Psen1-2 KD versus a rescue experiment showing transduction with WT or P486A GFP-Presenilin-2 (as shown in A), immunolabeled with antibody against K10 or filaggrin. H2B-RFP marks regions transduced with the Psen1,2 shRNA LV. (D) Control or Psen1-2 KD epidermis immunolabeled with antibodies against E-cadherin or NICD 1 or counterstained with DAPI. Merged image shows NICD/ECAD overlay. (E) Images of differentiating keratinocytes cultured in 2 mM Ca²⁺ and immunolabeled with antibody against K10 (green). H2B-RFP (red nuclei) marks regions transduced with Psen1-2 shRNA and either WT GFP-Presenilin-2 or P486A GFP-Presenilin-2. (F) Quantification of K10 expression in differentiating keratinocytes transduced with Psen1-2 shRNA alone or rescued with WT or P486A Presenilin-2 GFP. Data in histogram are normalized to scrambled control and represent data from two independent experiments. (G) Quantification of NICD (Notch intracellular domain) and H2B-RFP colocalization in E17.5 epidermis from embryos transduced with Psen1,2 shRNA alone or with either WT or P486A Presenilin-2 GFP. The percentage of total nuclear area in suprabasal cells expressing H2B-RFP was measured and compared with the percentage of total nuclear area NICD expression (see Materials and methods). Bars: (A–D) 30 µm; (E) 15 µm.