Notch-induced nuclear accumulation of cyclin D1 in NRVCs is not caused by inhibition of export. (A) NRVCs were treated for 36 h with the GSK3β inhibitor BIO or control MetBIO before staining with MF20 and analysis of DNA content of cardiomyocytes (MF20⁺ population) by flow cytometry, as for Fig. 1. One of two experiments with identical outcomes is shown. (B–G) NRVCs were treated for 24 h with the indicated concentrations of BIO (B, C, E, and F) or MetBIO (D and G) before staining with MF20 (red, Alexa 594) and for cyclin D1 (green, Alexa 488; B–D) or phospho-Rb (Ser807/811; green, Alexa 488; E–G). BIO induced both nuclear accumulation of cyclin D1 and phosphorylation of Rb. (H) Ad-N2_ICD–infected or control NRVCs were transfected with 0.6 μg pTopFlash-tk-Luc or pGL3-tk-Luc, as indicated, and 0.2 μg of pGL3-Renilla-Luc before exposure with BIO (0.25 μM, 0.5 μM, or 1 μM) for an additional 36 h before luciferase activities were determined. Firefly luciferase activity was normalized using Renilla luciferase activity. N2_ICD did not modulate the β-catenin/T cell factor–dependent transcription. Error bars indicate ±SD. (I) Total and phospho–Thr₂₈₆–cyclin D1 in NRVCs infected with Ad-N2_ICD or treated with 10% FCS or PHE (20 μM). N2_ICD did not prevent Thr₂₈₆ phosphorylation of cyclin D1. (J) LMB enhanced nuclear localization of cyclin D1 (green, Alexa 488) by N2_ICD (red, Alexa 594; arrows) but did not act on uninfected cells (circled nuclei), which is consistent with N2_ICD regulating nuclear localization upstream of export by the CRM1–exportin-1 complex. Bars: (B–D) 10 μm; (E–G) 25 μm; (J) 10 μm.