Supplemental Figure S1: effect of DHA on migration and proliferation of VSMC.

Panel A: After 24 hours of serum starvation cells were pre treated two hours with or without 20 µM of mitomycin C. Then a linear wound was made in the center of culture dish and cells were treated by IL-1 (10 ng/ml) and/or DHA (50µM) during48 hours (t48). The surface of the denuded zone was calculated as described in the methods. For each condition, the results are expressed as % of colonized zone at t0 and represent mean ± SE for 4 independent experiments.

Panel B: VSMC (105cells/well) were incubated with or without IL-1 in presence or absence of DHA for 48 hours. 5-bromo-2’deoxyuridine (BrdU) was added to the medium overnight. and BrdU incorporation was measured according to the manufacturer instructions. For each condition, the results are expressed as optic density and represent mean ± SE for 4 independent experiments. Similar results were observed when cells are seeded at 104 cells/well.

*p<0.01, ***p<0.0001, vs control and †p<0.01, ††p<0.001, †††p<0.0001 IL-1Dvs IL-1

Supplemental Figure S2: effect of DHA on the MMPs inhibitors TIMP-1 and TIMP-2.

After 48 hours of IL-1 (10ng/ml) and/or DHA (50µM) mRNA were extracted as described under methods. The mRNA level of TIMP-1 and TIMP-2 were determined by real time RT-PCR (Light Cycler, Roche). Amounts of mRNA expression were systematically normalized to HPRT transcripts level. **p<0.001, ***p<0.0001 vs control; ns: no significant, ††p<0.001 vs IL-1.

Supplemental Figure S3: 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. Panel A: western blot using antibodies against Notch Val1744 and β-actin.

Panel 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 S4: effect of DAPT on migration and proliferation of VSMC.

Panel A: After 24 hours of serum starvation cells were pre treated two hours with or without 20 µM of mitomycin C. Then a linear wound was made in the center of culture dish and cells were treated by IL-1 (10 ng/ml) and/or DAPT (0.5 µM) during48 hours (t48). The surface of the denuded zone was calculated as described in the methods. For each condition, the results are expressed as % of colonized zone at t0 and represent mean ± SE for 4 independent experiments.

Panel B: VSMC (105cells/well) were incubated with or without IL-1 in presence or absence of DAPT for 48 hours. 5-bromo-2’deoxyuridine (BrdU) was added to the medium overnight. and BrdU incorporation was measured according to the manufacturer instructions. For each condition, the results are expressed as optic density and represent mean ± SE for 4 independent experiments. Similar results were observed when cells are seeded at 104 cells/well. *p<0.01, **p<0.001, ***p<0.0001, vs control; ns: not significant vs IL-1.

Supplemental Figure S5: effect of DAPT on the MMPs inhibitors TIMP-1 and TIMP-2.

After 48 hours of IL-1 (10ng/ml) and/or DAPT (0.5 µM) mRNA were extracted as described under methods. The mRNA level of TIMP-1 and TIMP-2 were determined by real time RT-PCR (Light Cycler, Roche). Amounts of mRNA expression were systematically normalized to HPRT transcripts level. **p<0.001, vs control; ns: no significant, †p<0.01 vs IL-1.