Upregulation of Dickkopf1 by Oscillatory Shear Stress Accelerates Atherogenesis

ELECTRONIC SUPPLEMENTARY MATERIAL ESM, J Mol Med

Upregulation of Dickkopf1 by oscillatory shear stress accelerates atherogenesis

Mengmeng Li, Xinxin Liu, Yu Zhang, Mingxue Di, Han Wang, Lin Wang, Yifei Chen, Xiaoling Liu, Xiaoqing Cao, Renya Zeng, Yun Zhang, Mei Zhang#

The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital of Shandong University, Jinan, China, 250012

Running title: DKK1 and atherosclerosis

# Send Correspondence and Reprint Requests to: Mei Zhang Department of Cardiology Qilu Hospital, Shandong University 107#, Wen Hua Xi Road, Jinan, Shandong, 250012, P.R. China Phone: 86-0531-82169139 Fax: 86-0531-86169356 E-mail: [email protected]

1 Table S1. Antibodies used in this study

Primary Host Dilution and supplier Application antibodies DKK1 Mouse 1:500 for WB, 1:50 for IF, 1:200 for IHC; WB, IF, Abcam, Cambridge, MA IHC CD31 Rat 1:50; Santa Cruz, Dallas, TX IF VCAM1 Rabbit 1:1000; Abcam, Cambridge, MA WB ICAM1 Rabbit 1:1000; Abcam, Cambridge, MA WB E-selectin Rabbit 1:500; ProteinTech Group, Chicago, IL WB ZO1 Rabbit 1:1000 for WB, 1:50 for IF; ProteinTech WB, IF Group, Chicago, IL ZO2 Rabbit 1:1000 for WB, 1:100 for IF; Cell Signaling, WB, IF Danvers, MA Occludin Rabbit 1:1000 for WB, 1:100 for IF; Abcam, WB, IF Cambridge, MA Claudin 5 Rabbit 1:500 for WB, 1:50 for IF; Abcam, WB, IF Cambridge, MA MOMA-2 Rat 1:50; Abcam, Cambridge, MA IF PAR1 Rabbit 1:300; Abcam, Cambridge, MA WB CREB Rabbit 1:1000; Cell Signaling, Danvers, MA WB p-CREB (Ser 133) Rabbit 1:1000; Abcam, Cambridge, MA WB α-SMA Rabbit 1:100; Abcam, Cambridge, MA IHC

β-catenin Rabbit 1:1000; Cell Signaling, Danvers, MA WB

p-β-catenin Rabbit 1:1000; Cell Signaling, Danvers, MA WB

Table S2. Primer pairs of the target genes used for real time RT-PCR in this study

Genes Forward Reverse Homo DKK1 GGGAATTACTGCAAAAATGGAATA ATGACCGGAGACAAACCAGAAC Homo PAR1 GTTGATCGTTTCCACGGTCT ACGCAGAGGAGGTAAGCAAA

2 Homo β-actin CGTGCGTGACATTAAGGAGA CACCTTCACCGTTCCAGTTT Mus DKK1 CTCATCAATTCCAACGCGATCA GCCCTCATAGAGAACTCCCG Mus β-actin GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT

3 Fig. S1 Partial ligation of left common carotid artery (LCA) caused disturbed flow in mice. (a) Ultrasound images showing flow velocity profiles pre-ligation and 1 day post- ligation and revealing that partial ligation induced flow reversal (indicated by red arrows) in the LCA during diastole. Flow in the right common carotid artery (RCA) remained unchanged after ligation. (b) Partial ligation reduced blood flow through the LCA, without significantly raising flow in RCA. * P<0.05 vs. pre-ligated mice (n=8). (c) Western blot analysis of protein levels of mechanosensitive proteins VCAM1 and ICAM1 at 2 days after ligation in LCA of sham and ligated groups. * P<0.05 vs. sham-operated mice (n=8).

4 Fig. S2 In vivo gene silencing of DKK1 by caudal vein lentiviral gene delivery in

ApoE-/- mice. (a) Representative fluorescent photomicrographs for the left common carotid artery 2 weeks after pGLV3-shRNA-DKK1 delivered into mice. (b) Immunostaining of the left common carotid artery 2 weeks after pGLV3-shRNA-DKK1 delivered into mice, showing the efficiency of lentiviral delivery.

5 Fig. S3 DKK1 expression in vascular smooth muscle cells (VSMC) under disturbed flow showed no significant change. (a) Immunostaining of α-SMA (VSMC marker) and DKK1 on serial cross-sections. Black arrows refer to endothelium and black stars to VSMC. (b) Western blot analysis of DKK1 protein expression under oscillatory shear stress (OSS) for 6 h in HUVEC and VSMC. * P<0.05 vs static control (Con) (n=6).

6 Fig. S4 DKK1 negatively regulated Wnt/β-catenin pathway in vitro. Western blot analysis of the expression of phosphorylated and total β-catenin in HUVECs transfected with DKK1 siRNA. * P<0.05 vs. control without treatment and # P<0.05 vs. only OSS treatment (n=6).