Von Hippel-Lindau Protein Is Required for Optimal

0 downloads 0 Views 848KB Size Report
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

Suggest Documents