Figure 4. Notch signaling represses Sox9 expression to allow acquisition and maintenance of vSMC contractile phenotype.

A- Transcriptional level of Sox9 and Cnn1 was determined by qRT-PCR in WT MEFs and E14.5 vSMC, normalized and represented relative to Hprt housekeeping gene. n=8–12; B-Sox9 transcript level was evaluated by qRT-PCR from dorsal aortae harvested at the indicated stages and represented relative to Hprt. J1^WT (black bars), heterozygous (J1^SMHTZ, blue bars) and J1^SMKO (red bars) DA were analyzed. Green dotted line represents the trend of expression of J1^WT over time. nJ1^WT= 5–9; n J1^SMHTZ =3–5; nJ1^SMKO=3–5. C- Immunodetection of Sox9 (green; LUT) in combination with αSMA (red) in the DA at E12.5 and E14.5. Bottom panels are higher magnification view of respective top panels. Scale Bars: 25µm. D–E- Protein level of Sox9 in the cytoplasm (C) and in the nucleus (N) of J1^WT and J1^SMKO E14.5 vSMC was quantified by Western blot. GAPDH and LAMIN A/C were used as loading control for cytoplasmic and nuclear fraction respectively. n=3 (E). F- WT vSMC at E14.5 were treated with siRNA targeting Sox9 previous to DAPT treatment (50µM- 24h). Transcriptional analysis of Acta2, Tagln, Cnn1 and Myh11 was performed by qRT-PCR and represented as fold change compared to control treated with scramble siRNA and DMSO. Data are represented as mean +/− SEM n=7. A,B,E,F: *p<0.05; **p<0.001; ***p<0.0005; ****p<0.0001. E- Constant pressure of Notch signaling through Jag1 in the SM22⁺ compartment represses Sox9 expression in vivo. This prevents reprogramming of immature vSMC and allows maturation toward a contractile phenotype.