Fig. 6

Activation of Notch signaling rescues self-renewal in C/EBPβ-deficient myoblasts. a Western analysis of C/EBPβ and NICD expression in primary myoblasts isolated from C/EBPβ conditional knockout mice (β^−/−) or wild-type non-Cre-expressing littermates (WT) subjected to in vitro 4′OH-TAM treatment and cultured in growth medium (GM). Cyclophilin B (CyPB) is a loading control. b RT-qPCR analysis of Hey1 and Heyl expression in primary myoblasts isolated and cultured as in a (n = 5). c C/EBPβ and Notch1 intracellular domain (NICD) protein expression in wild-type (WT) and C/EBPβ^−/− (cKO) primary myoblasts retrovirally transduced to express NICD or with empty virus (pLPCX; pLP) cultured in GM for 48 h (n = 3). Percentage of d self-renewing (Pax7⁺/MyoD⁻), e proliferating (Pax7⁺/MyoD⁺), and f differentiating (Pax7⁻/MyoD⁺) WT (black bars), WT + NICD (blue bars), C/EBPβ^−/− (white bars), and C/EBPβ^−/− + NICD (orange bars) myoblasts cultured as in c as determined by immunocytochemistry (n = 3). g Percentage of myogenin⁺ cells relative to total nuclei (n = 3). h RT-qPCR analysis of Notch1, Notch2, and Notch3 expression in primary cells transduced to express C/EBPβ and cultured as in a, shown relative to WT controls indicated by the red line (n = 5). i Cebpb and j Notch1, Notch2 and Notch3 expression in C2C12s retrovirally transduced to express C/EBPβ relative to empty vector controls (pLXSN, red line) cultured in growth medium for 24 h (n = 4). k ChIP analysis of C/EBPβ occupancy at four regions (R1–R4) of the Notch2 locus in C2C12s retrovirally transduced to express C/EBPβ or empty vector (pLXSN) in GM. RT-qPCR data is shown as the percent enrichment relative to 10% input used for immunoprecipitation for each condition (n = 4). Approximate region locations are relative to the transcriptional start site. Data is represented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. Means with different letters are significantly different