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SUPPLEMENTARY DATASET

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PPARγγγγ COUNTERACTS LRP1-INDUCED VASCULAR CALCIFICATION BY INHIBITING

WNT5A SIGNALING IN VASCULAR SMOOTH MUSCLE CELL

Estelle Woldt1, Jérome Terrand1, Mohamed Mlih1, Rachel L. Matz1, Véronique

Bruban1, Fanny Coudane1, Sophie Foppolo1, Zeina El Asmar1, Maria Eugenia Chollet2, Ewa Ninio2, Audrey Bednarczyk3, Danièle Thiersé3, Christine Schaeffer3, Alain Van

Dorsselaer3, Christian Boudier1, Walter Wahli4, Pierre Chambon5, Daniel Metzger5, Joachim Herz6, Philippe Boucher1

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Supplementary Figure S1: Vascular calcification in male and female mice

Supplementary Figure S1 (a) Opened and Sudan IV-stained aortas from smPPARγ- female

mice and controls (smPPARγ+). Arrowheads show lipid-laden (Sudan positive)

atherosclerotic lesions. Scale bar, 0,5 cm. (b) Atherosclerotic lesion size in aortas from female

smPPARγ- (n=4) and controls (n=5) mice. (c) Histological analysis of calcified

atherosclerotic lesions in aortas from male and female smPPARγ- mice. Scale bar: 50 µm. (d)

Von Kossa staining of chondrocyte-like cells in aortas from control mice (smPPARγ+). Scale

bar: 50 µm. (e) RT-PCR analysis of the indicated genes in aortas from male (n=3) and female

(n=3) smPPARγ- and control mice (n=6) fed an atherogenic diet. Error bars, s.d.*, p<0,05. **,

p<0,01.

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Supplementary Figure S2: Absence of PPARγγγγ accelerates chondrogenesis

Supplementary Figure S2 (a) Alizarin red and alcian blue staining of primary cultured

PPARγ-/- and controls skin fibroblast treated with a chondrogenic cocktail for 15 days (n=4).

(b) Microscopical analysis of primary cultured vSMCs PPARγ-/- and controls treated with a

chondrogenic cocktail for 15 days and stained with alcian blue. Scale bar, 20 µm. (c) The

Sox9 (-2 kb) and the Cart1 promoter is linked to a luciferase reporter. (d) sFRP1 mRNA

levels in aortas from smPPARγ- and control mice (n=5). (e) Fra2 and (f) Osteocalcin mRNA

levels in aortas from smPPARγ- (n=5) and control (n=5) mice fed an atherogenic diet and

treated with rosigitazone for 2months. Error bars, s.d.*, p<0,05.

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Supplementary Figure S3: LRP1 is required for chondrogenesis:

Supplementary Figure S3: (a) Identification of PPREs in the sFRP2 promoter. (b) Alkaline

phosphatase and (c) sFRP2 mRNA levels in aortas from smLRP1-, (n=4) and control (n=4)

mice fed an atherogenic diet. (d) Alizarin red and alcian blue staining, and (e) Sox9 mRNA

levels in primary cultured LRP1-/- (n=4) and control (n=4) (LRP1+/+) skin fibroblast treated

with a chondrogenic cocktail for 15 days. (f) Alkaline phosphatase mRNA levels in aortas

from smLRP1-/smPPARγ- (n=5), and control (n=5) mice fed an atherogenic diet. (g) Alizarin

red staining of cultured vSMCs isolated from aortas of smLRP1-/smPPARγ- (n=5) and

control mice (n=5), and treated with a chondrogenic cocktail for 15 days. (h) Sox9 and Cart1

mRNA levels in cultured vSMCs isolated from aortas of smLRP1-/smPPARγ- (n=4) and

control (n=4) mice, and treated with a chondrogenic cocktail for 15 days. Error bars, s.d.*,

p<0,05.