Fig. 3. Notch stimulation expands Pax7-positive cells during myogenic differentiation in vitro. (A) Primary myoblasts were infected with retroviruses containing constitutively active mouse Notch-1 (caNotch-AP) or with control retroviruses (c-AP). One day after infection, cells were transferred to differentiation medium and, 1 day later, cells were fixed, co-stained with mouse anti-Pax7 and rabbit anti-cleaved Notch-1 (caNotch-AP-infected cells) or anti-Flag antibodies (c-AP-infected cells) and analyzed by immunofluorescence microscopy. The relative number of Pax7-positive cells among NICD-negative and NICD-positive cells (or Flag-negative and Flag-positive cells) on the same slide was calculated (mean ± s.e.m.; n=3; at least 200 Pax7-positive cells were counted in each determination). (B) Primary myoblasts or C2C12 cells (70% confluent) were co-cultured for 1 day with CHO cells stably transfected with mouse DLL1 (CHO-DLL1) or with empty vector (CHO-V). The levels of NICD, Pax7, MyoD, myogenin, p21 and tubulin were analyzed by western blotting.