Supplemental figure legends Figure S1. H&E staining ... - cloudfront.net
Supplemental figure legends
Figure S1. H&E staining of heart sections of E14.5 embryos.
S259A embryos have much thinner ventricle wall (arrow) increased trabeculation (arrowhead). Figure S2. RAf1S259A does not affect LEC proliferation.
LEC proliferation in E12.5 (A) and E14.5 (D) embryos was assessed by Ki67 (green) and PROX1 (red) immunofluorescence staining. PROX1+ and PROX1+/Ki67+ cells were counted from fifteen consecutive 7-μm sections of each embryo at the indicated positions as shown in (A) and (D). The values were then averaged to represent the mean number for each embryo. Control, n=3 embryos; S259A, n=3 embryos. Data represents Mean of mean±SEM. LEC proliferation was calculated as percentage of PROX1+/Ki67+ cells (B, E) versus total PROX1 positive cells (C, F). No significant difference in LEC proliferation was found. ls, lymph sac. Scale bar: 36 μm. Figure S3. RAF1S259A induces PROX1.
PROX1 (red) and VEGFR3 (cyan) immunofluorescence staining of E14.5 embryo sections. PROX1 was still detectable in the cardinal vein of S259A embryos (arrowheads) but not in that of control wild type embryos. Scale bar: 200 μm.
Figure S4. SOX18 expression in S259A cells at EE11.5 and E14.5. SOX18 (green) and β-GAL (purple) immunofluorescence staining of E11.5 and E14.5 embryo sections. Nuclei are labeled with DAPI (blue). Arrow heads indicate SOX18-expressing cells. jv, jugular vein, jls, jugular lymph sac. Scale bar: 25 μm.
34
Figure S5. RAF1S259A does not affect COUP-TFII expression.
(A) Immunofluorescence staining of COUP-TFII (green) revealed no difference in COUP-TFII expression between S259A and control embryos in either jugular veins or lymph sacs at E12.5. (B) Immunofluorescence staining of COUP-TFII (red) and SOX18 revealed no difference in COUP-TFII expression between S259A and control embryos in either jugular veins or lymph sacs at E14.5, while SOX18 level was higher in S259A embryos compared to the control embryos. cv, cardinal vein; jv, jugular vein; jls, jugular lymph sac. (C) COUP-TFII expression in HUVECs infected with empty control, RAF1 WT and S259A lentiviruses was analyzed by qPCR. Data are means ±s.e. of three replicates. Scale bar: 25 μm.
Figure S6. Inhibition of ERK signaling reduces PROX1+ cells. (A) Immunofluorescence staining of PROX1 (green), β-Gal (magenta) and DAPI (blue) of DMSO or U0126 treated E12.5 embryo sections. cv, cardinal vein. Scale bar: 200 μm. (B) Quantification of PROX1+ cells was performed by counting PROX1+ cells in each field as shown in (A). Control, n=4 embryos; S259A, n=4 embryos. Mean±SEM.
Figure S7. ERK regulation of lymphatic fate
A proposed scheme of ERK-dependent regulation of PROX1 expression. ERK signaling regulates lymphatic endothelial cell fate by controlling SOX18 and PROX1 expression.
35
Table S1. List of primers used for qPCR.
SOX18 FWR
CTTCATGGTGTGGGCAAAG
SOX18 REV
GCGTTCAGCTCCTTCCAC
SOX7 FWR
CAGCAAGATGCTGGGAAAGT
SOX7 REV
GTTGGGGTAGTCCTGCATGT
GAPDH FWR
TGCACCACCAACTGCTTAGC
GAPDH REV
GGCATGGACTGTGGTCATGAG
COUP-TFII FWR
GCAAGTGGAGAAGCTCAAGG
COUP-TFII REV
GCTTTCCACATGGGCTACAT
LYVE1 FWR
ACTTCCATCTGGACCACGAG
LYVE1 REV
AGCCTACAGGCCTCCTTAGC
SOX17 FWR
CAGAATCCAGACCTGCACAA
SOX17 REV
GCGGCCGGTACTTGTAGTT
PROX1 FWR
GGCATTGAAAAACTCCCGTA
PROX1 REV
ACAGGGCTCTGAACATGCAC
VEGFR3 FWR
GGTGTCGATGACGTGTGACT
VEGFR3 REV
CTCTGCCTGGGACTCCTG
PDPN FWR
CGAAAAATGTCGGGAAGGTA
PDPN REV
GGTCACCGTGGATTCTGAGT
Pecam1 FWR
AACAGAAACCCGTGGAGATG
Pecam1 REV
GTCTCTGTGGCTCTCGTTCC
Sox18 FWR
AACAAAATCCGGATCTGCAC
Sox18 REV
CGAGGCCGGTACTTGTAGTT
Vegfr3 FWR
GCTGTTGGTTGGAGAGAAGC
Vegfr3 REV
TGCTGGAGAGTTCTGTGTGG
Gapdh FWR
AACTTTGGCATTGTGGAAGG
Gapdh REV
ACACATTGGGGGTAGGAACA
36
S259A
20X
4X
Control
Figure S1
ls ls
E14.5 E14.5
50
40 NS
30
20
10 0
59
20 0
F 9A
40
Prox1+ cells per jugular lymphatic sac
NS
S2 5
l A
C on tr o l
S2
60
50
9A
E
C
S2 5
S259A
80
C on tr o l
D
E12.5
Prox1+ cells per jugular lymphatic sac
E12.5 on t ro
36μm
9A
l
Control
B
S2 5
C
ls
Ki67/PROX1 on t ro
ls Ki67/PROX1
ls
C
S259A Proliferating Prox1+ cells (%)
Control
Proliferating Prox1+ cells (%)
A E12.5 P = 0.0372
40
30
20 10 0
E14.5
40
30 P = 0.0287
20
10 0
Figure S2
Control jls
S259A jls jls jls
cv 200 μm
cv
cv
cv E14.5
Figure S3
S259A Control S259A Control
DAPI
Merge
cv jv
jv E14.5
S259A
cv
β-GAL
E11.5
Control
SOX18
jls
jls
Figure S4
Merge
B
cv
C ls
Merge
jv jls
S259A
cv
SOX18 Coup-TFII β-GAL
jv jls E14.5 Coup-TFII
mRNA per 10 Gapdh
β-GAL
Control
CD31
80 P=0.032
60
0
T S2 59 A
E12.5
20
W
ls
40
C on tro l
S259A
3
Control
S259A
Control
A COUP-TFII
Figure S5
A
DMSO
U0126
B
cv
200
100 50
U0126
S259A
DMSO
E12.5
U0126
cv
P
Figure S1. H&E staining of heart sections of E14.5 embryos.
S259A embryos have much thinner ventricle wall (arrow) increased trabeculation (arrowhead). Figure S2. RAf1S259A does not affect LEC proliferation.
LEC proliferation in E12.5 (A) and E14.5 (D) embryos was assessed by Ki67 (green) and PROX1 (red) immunofluorescence staining. PROX1+ and PROX1+/Ki67+ cells were counted from fifteen consecutive 7-μm sections of each embryo at the indicated positions as shown in (A) and (D). The values were then averaged to represent the mean number for each embryo. Control, n=3 embryos; S259A, n=3 embryos. Data represents Mean of mean±SEM. LEC proliferation was calculated as percentage of PROX1+/Ki67+ cells (B, E) versus total PROX1 positive cells (C, F). No significant difference in LEC proliferation was found. ls, lymph sac. Scale bar: 36 μm. Figure S3. RAF1S259A induces PROX1.
PROX1 (red) and VEGFR3 (cyan) immunofluorescence staining of E14.5 embryo sections. PROX1 was still detectable in the cardinal vein of S259A embryos (arrowheads) but not in that of control wild type embryos. Scale bar: 200 μm.
Figure S4. SOX18 expression in S259A cells at EE11.5 and E14.5. SOX18 (green) and β-GAL (purple) immunofluorescence staining of E11.5 and E14.5 embryo sections. Nuclei are labeled with DAPI (blue). Arrow heads indicate SOX18-expressing cells. jv, jugular vein, jls, jugular lymph sac. Scale bar: 25 μm.
34
Figure S5. RAF1S259A does not affect COUP-TFII expression.
(A) Immunofluorescence staining of COUP-TFII (green) revealed no difference in COUP-TFII expression between S259A and control embryos in either jugular veins or lymph sacs at E12.5. (B) Immunofluorescence staining of COUP-TFII (red) and SOX18 revealed no difference in COUP-TFII expression between S259A and control embryos in either jugular veins or lymph sacs at E14.5, while SOX18 level was higher in S259A embryos compared to the control embryos. cv, cardinal vein; jv, jugular vein; jls, jugular lymph sac. (C) COUP-TFII expression in HUVECs infected with empty control, RAF1 WT and S259A lentiviruses was analyzed by qPCR. Data are means ±s.e. of three replicates. Scale bar: 25 μm.
Figure S6. Inhibition of ERK signaling reduces PROX1+ cells. (A) Immunofluorescence staining of PROX1 (green), β-Gal (magenta) and DAPI (blue) of DMSO or U0126 treated E12.5 embryo sections. cv, cardinal vein. Scale bar: 200 μm. (B) Quantification of PROX1+ cells was performed by counting PROX1+ cells in each field as shown in (A). Control, n=4 embryos; S259A, n=4 embryos. Mean±SEM.
Figure S7. ERK regulation of lymphatic fate
A proposed scheme of ERK-dependent regulation of PROX1 expression. ERK signaling regulates lymphatic endothelial cell fate by controlling SOX18 and PROX1 expression.
35
Table S1. List of primers used for qPCR.
SOX18 FWR
CTTCATGGTGTGGGCAAAG
SOX18 REV
GCGTTCAGCTCCTTCCAC
SOX7 FWR
CAGCAAGATGCTGGGAAAGT
SOX7 REV
GTTGGGGTAGTCCTGCATGT
GAPDH FWR
TGCACCACCAACTGCTTAGC
GAPDH REV
GGCATGGACTGTGGTCATGAG
COUP-TFII FWR
GCAAGTGGAGAAGCTCAAGG
COUP-TFII REV
GCTTTCCACATGGGCTACAT
LYVE1 FWR
ACTTCCATCTGGACCACGAG
LYVE1 REV
AGCCTACAGGCCTCCTTAGC
SOX17 FWR
CAGAATCCAGACCTGCACAA
SOX17 REV
GCGGCCGGTACTTGTAGTT
PROX1 FWR
GGCATTGAAAAACTCCCGTA
PROX1 REV
ACAGGGCTCTGAACATGCAC
VEGFR3 FWR
GGTGTCGATGACGTGTGACT
VEGFR3 REV
CTCTGCCTGGGACTCCTG
PDPN FWR
CGAAAAATGTCGGGAAGGTA
PDPN REV
GGTCACCGTGGATTCTGAGT
Pecam1 FWR
AACAGAAACCCGTGGAGATG
Pecam1 REV
GTCTCTGTGGCTCTCGTTCC
Sox18 FWR
AACAAAATCCGGATCTGCAC
Sox18 REV
CGAGGCCGGTACTTGTAGTT
Vegfr3 FWR
GCTGTTGGTTGGAGAGAAGC
Vegfr3 REV
TGCTGGAGAGTTCTGTGTGG
Gapdh FWR
AACTTTGGCATTGTGGAAGG
Gapdh REV
ACACATTGGGGGTAGGAACA
36
S259A
20X
4X
Control
Figure S1
ls ls
E14.5 E14.5
50
40 NS
30
20
10 0
59
20 0
F 9A
40
Prox1+ cells per jugular lymphatic sac
NS
S2 5
l A
C on tr o l
S2
60
50
9A
E
C
S2 5
S259A
80
C on tr o l
D
E12.5
Prox1+ cells per jugular lymphatic sac
E12.5 on t ro
36μm
9A
l
Control
B
S2 5
C
ls
Ki67/PROX1 on t ro
ls Ki67/PROX1
ls
C
S259A Proliferating Prox1+ cells (%)
Control
Proliferating Prox1+ cells (%)
A E12.5 P = 0.0372
40
30
20 10 0
E14.5
40
30 P = 0.0287
20
10 0
Figure S2
Control jls
S259A jls jls jls
cv 200 μm
cv
cv
cv E14.5
Figure S3
S259A Control S259A Control
DAPI
Merge
cv jv
jv E14.5
S259A
cv
β-GAL
E11.5
Control
SOX18
jls
jls
Figure S4
Merge
B
cv
C ls
Merge
jv jls
S259A
cv
SOX18 Coup-TFII β-GAL
jv jls E14.5 Coup-TFII
mRNA per 10 Gapdh
β-GAL
Control
CD31
80 P=0.032
60
0
T S2 59 A
E12.5
20
W
ls
40
C on tro l
S259A
3
Control
S259A
Control
A COUP-TFII
Figure S5
A
DMSO
U0126
B
cv
200
100 50
U0126
S259A
DMSO
E12.5
U0126
cv
P
