Enforced expression of jagged1 interfered with cell cycle progression and antagonized IL-1β.

<p>(A) The non-dystrophic myogenic cell clone Hu37KD5 (white column) was transduced with a control lentivirus vector (Hu37CTR, gray column) or a Jagged1-expression lentivirus (Hu37rJ1, filled column). The amounts of transcripts of Jagged1, Notch1, Notch2, and Notch3 were determined by qRT-PCR. The amounts of mRNA were normalized to control the POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. (B-C) Twenty micrograms of total proteins from Hu37CTR (CTR) and Hu37rJ1 (J1) were subjected to immunoblotting with antibodies against Jagged1 (B) and Notch3 (C). F, Unprocessed full-length protein; CTF, cytoplasmic fragment; asterisks, non-specific bands. Numbers represent the positions of protein size markers. (D) Hu37CTR (white circles) and Hu37rJ1 (filed circles) cell clones were treated with IL-1β (0–250 pg/ml) for 24 h. The cells were incubated with EdU (10 μM) for a further 6 h culture, and then subjected to fluorescence analysis. EdU-positive nuclei were counted in nine areas of each culture and the means and standard deviation were estimated. Data were statistically analyzed using one-way ANOVA. The statistical significance of the difference between the unstimulated control (0 pg/ml) and cytokine-stimulated cells (60–250 pg/ml) was less than 0.01 in both Hu37CTR and Hu37rJ1. The statistical significance of the difference between Hu37CTR and Hu37rJ1 was less than 0.01 at each dosage of IL-1β. (E) The expression of CSF2 in Hu37CTR (white column) and Hu37rJ1 (gray column) cells was analyzed by qRT-PCR after 24 h of exposure to IL-1β. The amounts of mRNA were normalized to the control POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. Data were statistically analyzed using Student’s <i>t</i>-test. *, p<0.05.