Notch3 and IL-1 exert opposing effects on a vascular smooth muscle cell inflammatory pathway in which NF-B drives crosstalk

JCS007872 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression and functionality of Delta1-Fc Notch ligand in vascular smooth muscle cells. (A) 50 μl of conditioned medium collected from wild-type HEK 293T cells (WT CM) or HEK 293T cells transfected with a vector encoding a fusion protein including the extracellular domain of rat Delta1 and Fc fragment of human IgG (Δ1-Fc CM) were separated by 10 % SDS PAGE. Expression of the chimeric protein Delta1-Fc was evidenced by immunoblotting using a rabbit anti-human IgG against the Fc fragment (Jackson ImmunoResearch, diluted 1:20,000). (B) Smooth muscle cells were incubated with WT CM or Δ1-Fc CM for 24 hours. Hes1, HRT1 and HRT2 transcripts were assayed by RT-PCR. Results are expressed as a percentage of the mRNA level from WT-CM-incubated cells and represent the mean of ± s.e.m. of three independent experiments. *P<0.05; **P<0.01; ns, non-significant.

• Supplemental Figure S2 -

  Fig. S2. Overexpression and functionality of Notch3 or Notch1 intracellular domains (N3-ICD or N1-ICD) in vascular smooth muscle cells. Cells were transfected with N3-ICD, N1-ICD or empty (mock) plasmids. (A) Left panel: gel migration of N3-ICD and N1-ICD PCR products; right panel: western blot using antibodies against Notch Val1744 and β-actin. (B) Transcripts encoding Hes1, HRT1 and HRT2 were assayed by RT-PCR. Results are expressed as a percentage of the mRNA level from mock-transfected cells. Values represent the mean ± s.e. of three independent experiments. N1-ICD or N3-ICD versus mock; *P<0.05; ns, non-significant.

• Supplemental Figure S3 -

  Fig. S3. Overexpression of the RBP-Jκ mutant in vascular smooth muscle cells. Cells were transfected with RBP-Jκ DN or empty (mock) plasmids. Upper panel: gel migration of the RBP-Jκ DN PCR product; lower panel: western blot using antibodies against the FLAG epitope and GAPDH.

• Supplemental Figure S4 -

  Fig. S4. Functionality of IκBα_32-36 mutant in IL-1β-treated vascular smooth muscle cells. Cells were transfected with IκBα_32-36 or empty (mock) plasmids and treated for 24 hours with IL-1β (10 ng/ml) or vehicle (control). The secretion of PLA₂ was expressed as a percentage of that of control cells: 5.1±0.3 and 4.5±0.7 pmol/min/μg RNA for mock and IκBα_32-36 transfected cells, respectively. **P<0.01.

• Supplemental Figure S5 -

  Fig. S5. Effect of DAPT on PLA₂ and PGE₂ secretion in IL-1β-pretreated cells. Cells were pretreated for 48 hours with IL-1? (10 ng/ml) or vehicle (control) and then DAPT (0.5 μM) was added for 24 hours. The secretion of PLA₂ and PGE₂ was expressed as a percentage of that of control cells (3.9±0.6 pmol/min/μg RNA and 7.41±2.29 pg/ml, respectively). The data represent the mean ± s.e.m. of three independent experiments. *P<0.05; ns, non-significant.