Extended Data Fig. 2: Sphk1 is essential for elastin deficiency-induced SMC hyperproliferation.
From: Sphingosine kinase 1 is integral for elastin deficiency-induced arterial hypermuscularization
Aortic SMCs were isolated from Eln(+/+), Eln(+/−) or Eln(−/−) pups at P0.5. (a-c) SMCs were cultured for 8 h either with EdU (a, b) and stained for EdU and nuclei (Hoechst), n = 4–6 (n = 5 Eln(+/+), n = 4 Eln(+/−), n = 6 Eln(−/−)) or with BrdU (c) and subjected to BrdU ELISA, n = 40 per group, biological replicates. (d) SMCs were treated with scrambled (Scr) RNA or siSphk1 and then subjected to qRT–PCR. n = 4 per group, biological replicates. (e, f) SMCs treated with Scr RNA or siSphk1 were cultured with EdU for 8 h and then stained for EdU and nuclei (Hoechst). n = 5 per group, biological replicates. (g, h) SMCs were treated with siRNA and then subjected to scratch assay. Percent of wound area relative to time 0 was quantified. n = 5 per group, biological replicates. (i-k) SMCs were treated with siRNA and then transfected with vector control or vector containing wild-type Sphk1 (Sphk1-WT) or catalytically inactive Sphk1 (Sphk1-CI). These SMCs were subjected to qRT–PCR for Sphk1 (i) and EdU cell proliferation assay (j, k). In i, n = 10 SMCs with Scr RNA and vector; n = 5 SMCs with Scr RNA and Sphk1-WT; n = 8 SMCs with Scr RNA and Sphk1-CI; n = 8 SMCs with siSphk1 and vector; n = 5 SMCs with siSphk1and Sphk1-WT; n = 7 SMCs with siSphk1and Sphk1-CI, biological replicates. In j, n = 9 per group, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test (b-d, f, i, k) or two-tailed unpaired Student’s t test (h). Scale bars, 100 μm.
