Fig. S3 .

 S3 Fig. S3. Morpholino design for Tspan18 targeting in zebrafish. (A) Genomic DNA organization of zebrafish Tspan18 shows exons (gray rectangles) and introns (solid lines). Location of targeting sites of ATGMO (green rectangle) and E7MO (red rectangle), and primer set flanking exon 7 (red arrows) are shown. (B) E7MO treated embryos show a smaller PCR product (using primers shown in (A), indicating deletion of exon 7 compared to E7MO∆. 


Fig. S4 .

 S4 Fig. S4. Quantitative RT-PCR analysis of VEGF/Notch/Semaphorin pathway components of 27hpf zebrafish injected with E7MO or E7MO. Gene expression levels (N=5) were normalized to β-actin and compared to that of E7MO∆ injected fish (set as 1.0). Error bars represent SD. P value was calculated using two-tailed Student's t-test. *: P<0.02; **: P<0.005. 


Fig. S5 .

 S5 Fig. S5. Knockdown of Tspan18 by siRNA in vitro. (A) The dose-dependent knockdown of Tspan18 by siRNA was confirmed in HUAEC by quantitative RT-PCR. Tspan18 siRNAΔ (20 nM) and transfection reagents only (0 nM) served as controls. Expression level was normalized to -actin. (B) The dosedependent knockdown of Tspan18 by siRNA was confirmed in HEK293T cells stably expressing hTspan18-myc protein by western blot analysis. Tspan18 siRNAΔ (20 nM) and transfection reagent only (0 nM) served as negative controls. The quantification was performed with ImageJ (NIH) and normalized to PBS sample. (C) Tspan18 knockdown by siRNA (20 nM) was confirmed with quantitative PCR in 211H and Hela cell. Tspan18 siRNAΔ was used as a negative control. 


Fig. S6 .

 S6 Fig. S6. Tspan18 is up-regulated in skin wound healing. (A) Quantitative RT-PCR of Tspan18 level during skin wound healing. (B) Immunofluorescent staining of Tspan18 (red), CD31 (green), and DAPI (blue) of skin wounds. 




  


Table S1 . Real-time RT-PCR primer sequences

 S1 
	Gene	Forward Primer (5'-3')	Reverse Primer (5'-3')
	hTspan18	TGGAGCACCATGGAAGGCGACTG	TGACCGAGTTCCAGGTGGCAGAG
	hTspan12	ATCGCTGCCGTTTTGCCCTTGGG	GCAATCATGACCGGATGAACCAC
	hVEGFa	ACCTCCACCATGCCAAGTGGTCC	TGCCCCTTTCCCTTTCCTCGAAC
	hVEGFR1	TCGCGCTCACCATGGTCAGCTAC	CAGGGAATGACGAGCTCCCTTCC
	hVEGFR2	ATGCAGAGCAAGGTGCTGCTGG	GACCGAGGCCAACTGAGTTTCC
	hVEGFR3	CACGGAGGAGTCACACGTCATCG	TGCGAGCGCAGCGTGACATTGAG
	hNotch1	GGTGAGACCTGCCTGAATG	GTTGGGGTCCTGGCATC
	hNotch4	CAGCCCAGTGGGTATCTCTG	GTTGTGACAGGGTTGGGACT
	hDll4	AGGCCTGTTTTGTGACCAAG	GTGCAGGTGTAGCTTCGCT
	hHey1	TGGATCACCTGAAAATGCTG	CGAAATCCCAAACTCCGATA
	hHey2	TTTGAAGATGCTTCAGGCAA	GGCACTCTCGGAATCCTATG
	hEphrinB2	TCCGTGTGGAAGTACTGCTG	ACCAGTCCTTGTCCAGGTAGAA
	hEphB4	ATGCCCGTCATGATTCTCAC	ACGAGCTGGATGACTGTGAA
	hActb	GCACAGAGCCTCGCCTT	GTTGTCGACGACGAGCG
	zTspan18	TAGGGGTGGGAATCTGGGTGCTG	AACTGGATTCCCCGAAACAAACAC
	zVEGF-AA	AGAGTGCGTGCAAGACCCGAGAG	GGCCTGCATTCACACTTGGTGTG
	zVEGFR1	GAAGGTGCAGTCGGAGATCCCAG	GCATCTCGCCCTGTAACGTGTGC
	zVEGFR2	GAGCCTCGGGTCAATGCTGTTCC	TCTGAAGGTCTGGGGACCTGCTG
	zVEGFR3	GGTGCTCAACTGCACTGCACTGG	GTCCCACTGGAAATCCACACCAG
	zNotch3	TGGTTCGCTCTGTCAGCATCTGG	GACATTGTGTACGGGCACAGGGC
	zDll4	ATGGCAGCTTGGCTCACCTTTCTC	CCTGCCATCCTTCTCTGCATACAC
	zNrarpA	ATGAGCCAGGCGGATATATCGACG	TCACCGCGCGCCGGACGAGTACTTG
	zNrarpB	ATGAGTCAGGCGGACATGACCTGC	TCAGAGCGCGCTTGATGAGTATTTC
	zEphrinB2	ACTGCTCAACTGTGACAAGCCG	TGCGCCGAGTGCTTCTGATGAC
	zEphB4	AGTGGACGACACACTCTCGCTC	AGACGGATAGTGAGCGCGTGAGC
	zSema3ab	GAGATGAATGCAAGTGGGCCGGG	CCGGCCCATGAAGTCAGCTGATG
	zPlxnD1	ACCTGTACCAGCTGAACGCGACG	GATCTCCGGGGTGTTCTCGAAGC
	zNrp1a	GGAGTCTTCTGCCGATACGACCG	GAGAAATGGCTCCCTGTGTGCCG
	zNrp1b	TGACATGGAGCCGGACACAACGG	TCAGTCTGGAGCGCAGTCGAGAC

Biology Open (2020): doi:10.1242/bio.050096: Supplementary information Biology Open • Supplementary information