Nuclear localization of cyclin D1 is RBP-Jκ independent. (A) NMVCs were isolated from RBP-Jκ^Flox/Flox pups on the day of birth (P1) and treated as indicated until processing for immunostaining (the equivalent of P6). 10% FCS was added to induce cyclin D1 in the absence of RBP-Jk. (B–D) Confocal images of NMVCs showing nuclear localization of cyclin D1 (green, Alexa 568; “a” and “b” panels) overlapping with DAPI (blue; “a” panels). α-actinin cytosolic immunostaining identified cardiomyocytes, and nuclear V5 epitope immunostaining identified cells expressing transduced N2_ICD (both visualized with a far red [Alexa 680] secondary antibody and shown as red in “c” panels). EGFP (D, d) shows Cre-EGFP fusion protein expression. Closed arrows indicate examples of Cre-EGFP⁺, N2_ICD⁺ cells with nuclear localization of cyclin D1. Open arrows indicate the rare cells that expressed N2_ICD but did not express Cre-EGFP; note the prominent nuclear presence of cyclin D1. The asterisks indicate a cell that expressed Cre-EGFP but not N2_ICD; note that cyclin D1 was cytoplasmic and perinuclear. (E) RBP-Jκ protein expression 1 and 3 d after Ad-Cre-EGFP treatment (corresponding to P3 and P5), showing efficient depletion by Cre.