Nitric Oxide Signaling in the Cardiovascular System. MYC. CCNA2. CCNE2 ..... (KPL). Recombinant SP-D served as standard and was included in the kit.
Cell Reports, Volume 24
Supplemental Information
Von Hippel-Lindau Protein Is Required for Optimal Alveolar Macrophage Terminal Differentiation, Self-Renewal, and Function Helena M. Izquierdo, Paola Brandi, Manuel-José Gómez, Ruth Conde-Garrosa, Elena Priego, Michel Enamorado, Sarai Martínez-Cano, Iria Sánchez, Laura Conejero, Daniel Jimenez-Carretero, Silvia Martín-Puig, Martin Guilliams, and David Sancho
1 2 3 4
SUPPLEMENTAL INFORMATION Figures S1-S3 Supplemental Experimental Procedures Supplemental References
1 2
A
Parabiosis
Analysis
CD45.1 Control Vhlfl/fl 30 days
Blood Neutrophils & MonocytesLy6Chi Lung MonocytesLy6Chi BAL AM
Control Vhlfl/fl CD11c∆Vhl CD45.1 CD11c∆Vhl
B
C Neutrophils
Monocytes
Ly6Chi
Blood
50 Freq. CD45.1+ (%)
Control Vhlfl/fl 87.3 12.7
76.3 23.7
75.6 24.3
Ly6C
Ly6G
CD11c∆Vhl
CD45.1
D
40 30
ns
ns
Control Vhlfl/fl CD11c∆Vhl
20 10
90.8 9.22 0 Neutrophils
MonocytesLy6Chi
CD45.1
Lung MonocytesLy6Chi BAL AM
E
Control Vhlfl/fl 89.6 10.4
99.8 0.24
88.5 11.5 CD45.1
3 4 5 6 7 8 9 10 11 12 13 14
CD11c
Ly6C
CD11c∆Vhl
Freq. of CD45.1 (%)
30 20
ns
10
Control Vhlfl/fl CD11c∆Vhl
2 2
ns
1
99.2 0.76
0 CD45.1
Lung MonocytesLy6Chi
BAL AM
Figure S1. Circulating monocytes do not contribute to Vhl-deficient AM phenotype. Related to Figure 2. (A) Scheme of parabiosis: CD45.2 control Vhlfl/fl and CD11c∆Vhl mice were surgically joined to a CD45.1 mouse in parabiosis. Thirty days after surgery, CD45.2 parabionts were analyzed. (B, C) Representative flow cytometry plots (B) and frequencies of CD45.1+ cells (C) in neutrophils (gated on CD11b+Ly6G+) and Ly6Chi monocytes (gated on CD11b+Ly6Chi) from peripheral blood. (D, E) Representative flow cytometry plots (D) and frequencies of CD45.1+ cells (E) in lung Ly6Chi monocytes (gated on CD45+ Siglec-F- CD11b+ Ly6Chi) and BAL AMs (gated on Siglec-F+ CD11c+). (C, E) Frequencies shown as mean ± s.e.m. of a pool of three independent experiments performed, where each dot represents an individual mouse (n=10-14 per genotype): ns, not significant (unpaired Student t-test).
A
CD11c-cre- Vhlfl/fl (Control Vhlfl/fl) vs. CD11c-cre+ Vhlfl/fl (CD11c∆Vhl)
CD11c-cre+ Vhl+/+ (Control CD11c-cre+) vs. CD11c-cre+ Vhlfl/fl (CD11c∆Vhl)
638
2531
520
B STAT1 ESR1 CCNA2 CDKN1A E2F3 E2F2 CCNE2 MYC CCNE1 TFDP1 CDKN1B
TOPBP1 TP53 BARD1 RBBP8 RPA1 RFC5 CHEK1 RAD51 GADD45A FANCD2 BRCA2 BRIP1 BRCA1 FANCA RFC3 CHEK2 PLK1
CDC25C KIF23 CDC20 PLK3 PRC1 CDC7 CCNB2 CCNB1 PLK4 PLK2 PKMYT1 FBXO5 KIF11 CDC25A CDK1 PPM1J
Mitotic Roles of Polo-like Kinase Estrogen-mediated S phase Entry Role of BRCA1 in DNA Damage Response Type I Diabetes Mellitus Signaling LPS/IL-1 mediated inhibition of RXR function
LXR/RXR Activation eNOS Signaling
IL1A PON1 APOE LYZ ABCG1 FASN ABCA1 CYP51A1 NR1H3 ECHS1 TLR4 C3 NR1H2 PCYOX1 SREBF1 TF IL1RN ACACA PLTP HMGCR C4A/C4B TNF SCD TNFRSF1B FDFT1 NFKB2 NFKB1
Rel. expression (β-Actin) (x10-3)
C
Ccnb1 3
0.08
Rel. expression (β-Actin) (x10-3)
1.5
0.8
Rel. expression (β-Actin) (x10-2)
0.08
0 ***
0.06
0.6
0.05
Cd25c 0.2
*
Plk3 15
ns
1
2
0.1
0
0
0
0
0
Nr1h3 ***
Abcg1
Nr1h2 2
**
30
*
Apoe 15
Control Vhlfl/fl CD11c∆Vhl
Cdkn1a 0.2
0.3
3
**
0
Tp53 4
2.5
7.5
0
Chek2 2
**
0.1
0
Chek1
Z-score -3
3
Cdk1 3
1.5
Brca1
Log FC -3
0.4
D
1 2 3 4 5 6 7 8 9 10
NRF2-mediated Oxidative Stress Response
Ccnb2 3
1.5
0
Nitric Oxide Signaling in the Cardiovascular System Ceramide Signaling
**
ns
Pltp 20
**
Control Vhlfl/fl CD11c∆Vhl
2 1
15
7.5
10
0
0
0
0
1 0
Figure S2. VHL-deficient AMs have an altered gene expression profile. Related to Figures 3 and 4. (A) Venn diagram showing genes differentially expressed in CD11c∆Vhl AMs versus control Vhlfl/fl AMs or control CD11c-cre+. (B) Circular plot representing Ingenuity Pathway Analysis of differentially expressed genes from pathways with |Zscore| > 2 and -log (B-H p-value) >2.5 in VHL-deficient AMs (CD11c∆Vhl) versus control Vhlfl/fl AMs from BAL. Depicted genes correspond to cell-cycle-related (blue) and lipid-handling-related (red) pathways. (C,D) Quantitative PCR of selected differentially expressed genes related to cell cycle (C) and lipid sensing (D), depicted in blue and red, respectively, in Figure S2B. ns, not significant; *p-value
