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. 2017 Nov 2;2(21):e93764. doi: 10.1172/jci.insight.93764

Figure 4. NOTCH3 is necessary for HemSC mural cell differentiation.

Figure 4

(A–C) Assessment of NOTCH3 knockdown (N3KD) in HemSCs. (A) Relative NOTCH3 transcript levels in control HemSCs (Scr), and N3KD HemSCs determined by qRT-PCR and normalized by BACTIN. Representative data from 4 independent N3KD HemSC populations and matching Scr HemSC populations are presented. Error bars represent ± SD. *P < 0.007, Student’s t test. (B) Representative data of Western analysis from 2 independent N3KD and matching Scr HemSC lysates probed with antibodies against NOTCH3 or α-tubulin as a loading control. (C) Relative HEYL and HES1 transcript levels in Scr and N3KD HemSCs determined by qRT-PCR and normalized by BACTIN expression. Representative data presented from 3 independent HemSC populations done in duplicate. Error bars represent ± SD. **P < 0.0007, ***P < 0.05, Student’s t test. (D) Scr HemSCs (n = 3) or N3KD HemSCs (n = 3) cocultured with ECFCs in growth media were stained for αSMA at days 0, 5, and 9. Scale bars: 50 μm. (E) Day 3, 5, 7, and 9 lysates from Scr and N3KD HemSC/ECFC cocultures were subjected to Western analysis, and probed with antibodies against αSMA or α-tubulin as a loading control. Ratio of αSMA band intensity normalized by α-tubulin band intensity presented below. Representative data from 3 Scr and N3KD HemSC populations and experiments perform in duplicate for each HemSC population. αSMA, α smooth muscle actin; ECFC, endothelial colony-forming cell; HemSC, hemangioma stem cell; Scr, scrambled.