Extended Data Fig. 1: In aortic SMCs, Sphk1 levels are regulated by elastin gene dosage, and Sphk1 deletion does not induce apoptosis.
From: Sphingosine kinase 1 is integral for elastin deficiency-induced arterial hypermuscularization
(a) Aortic lysates from Eln(+/+), Eln(+/−) or Eln(−/−) embryos at E13.5 were subjected to qRT–PCR for Sphk1. n = 6 mice per group. (b) Aortic SMCs were isolated from Eln(+/+), Eln(+/−) or Eln(−/−) pups at P0.5 and subjected to immunostaining for markers of SMCs (SMA) and nuclei (DAPI). Scale bar, 100 μm. (c) Lysates from isolated aortic SMCs of indicated genotypes were subjected to qRT–PCR for Eln or Sphk1. For Eln mRNA, n = 7 per group, biological replicates. For Sphk1 mRNA, n = 6 Eln(+/+) SMCs, n = 4 Eln(+/−) SMCs, n = 6 Eln(−/−) SMCs, biological replicates. (d, e) Aortic lysates from Eln(+/+), Eln(+/−) or Eln(−/−) mice at P0.5 (d) or from human aortic SMCs treated with scrambled (Scr) RNA or siRNA targeting ELN (e) were subjected to qRT–PCR for SPHK2. In d, n = 4 Eln(+/+) mice, n = 5 Eln(+/−) mice, n = 5 Eln(−/−) mice. In e, n = 4 per group, biological replicates. (f-j) Dams pregnant with Sphk1(flox/flox) embryos also carrying Acta2-CreERT2 or no Cre were injected daily with tamoxifen from E13.5 to E17.5. In (f-h), aortas were harvested from pups at P0.5 and subjected to lysis after adventitia and EC removal. In (f), extracted genomic DNA was subjected to PCR with primers flanking Sphk1. The 120, 250, and 600 PCR products represent the wild-type, floxed, and deleted Sphk1 alleles, respectively. In (g), isolated RNA was reverse transcribed and subjected to qRT–PCR. Histogram represents Sphk1 transcript levels relative to Gapdh. n = 4 non-Cre mice, n = 6 Acta2-CreERT2 mice. In (h), Western blot was performed on aortic lysates with densitometry of protein bands relative to β-actin shown on right. n = 4 mice per group. (i) Transverse aortic sections from mice of indicated genotypes at P0.5 were stained for markers of apoptosis (cleaved caspase-3), SMCs (SMA), and nuclei (DAPI). Section of the thymus from Eln(+/+), non-Cre pup at P0.5 is a positive control for cleaved caspase-3 staining. Lu, lumen. Scale bar, 50 μm. (j) Percent of SMCs that are cleaved caspase-3+ from sections represented in i. n = 5 Eln(+/+) mice per group, n = 6 Eln(+/−) mice, n = 6 Eln(−/−) mice per group. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (e, g, h) or one-way ANOVA with Tukey’s post hoc test (a, c, d, j).
