advances.sciencemag.org/cgi/content/full/sciadv.abb6075/DC1

Supplementary Materials for

MBD2 serves as a viable target against pulmonary fibrosis by inhibiting macrophage M2 program

Yi Wang, Lei Zhang, Guo-Rao Wu, Qing Zhou, Huihui Yue, Li-Zong Rao, Ting Yuan, Biwen Mo, Fa-Xi Wang, Long-Min Chen, Fei Sun, Jia Song, Fei Xiong, Shu Zhang, Qilin Yu, Ping Yang,

Yongjian Xu, Jianping Zhao, Huilan Zhang*, Weining Xiong* and Cong-Yi Wang*

*Corresponding author. Email: wangcy@tjh.tjmu.edu.cn, xiongdoctor@qq.com, huilanz_76@163.com

Published 4 December 2020, Sci. Adv. 6, eabb6075 (2020) DOI: 10.1126/sciadv.abb6075

This PDF file includes:

Figs. S1 to S12 Table S1

Supplementary Materials

Supplementary Figure 1. Histochemical staining of MBD2 in lung tissues from control

subjects and IPF patients (indicated by black arrows). Images were captured at ×400

magnification.

Supplementary Figure 2. Genotyping results of flox allele from LyzM-Cre+-

Mbd2flox/flox and LyzM-Cre+-Mbd2flox/- mice (A) The flox allele was indicated by arrows.

(B) The band for the LyzM-Cre allele.

Supplementary Figure 3. The level of lung injury and M2 macrophage activation

between Mbd2-C and LyzM-Cre mice after BLM challenge. Images were captured at

×200 magnification.

Supplementary Figure 4. MBD2 overexpression rescued the defect of Mbd2-CKO

BMDMs following IL-4 induction. Results for the time-course Western blot analysis

of Arg 1 expression in IL-4-induced Mbd2-CKO BMDMs following Mock or Mbd2

lentiviral transduction. Arg 1, arginase 1. The data are represented as the mean ± SEM.

**, p< 0.01; and ***, p< 0.001.

Supplementary Figure 5. The number of macrophages in the broncho-alveolar lavage

fluid (BALF) of clodronate liposome-treated or liposome-treated mice.

Supplementary Figure 6. The severity of pulmonary fibrosis in clodronate

liposomes or liposomes treated Mbd2-CKO mice following BLM induction. (A)

Upper panel: representative results for HE, Sirius red and Masson staining. Lower

panel: a bar graph figure showing the semiquantitative Ashcroft scores for the severity

of fibrosis. (B) Levels of fibronectin and collagen I in the lungs. All images were

captured at ×200 magnification, and seven mice were included in each study group.

Col I, collagen I; Fib, fibronectin. The data are represented as the mean ± SD. **, p<

0.01; and ***, p< 0.001.

Supplementary Figure 7. The activation of PI3K/Akt signaling and DNA

methylation of the Ship promoter in IPF patients. (A) Analysis of PI3K/Akt signaling

in IPF patients. (B) Results for the bisulfite DNA sequencing analysis of the Ship

promoter in IPF patients. The data are represented as the mean ± SD. *, p< 0.05; and

***, p< 0.001.

Supplementary Figure 8. A diagram showing the interacted regions in Ship

promoter. Methylated CpG DNA in 5 regions (from -1336 bp to +167 bp, the

transcriptional start site as +1) was highlighted within the Ship promoter.

Supplementary Figure 9. Characteristics of the liposomes. (A) Preparation of

Mbd2 siRNA-loaded liposomes. (B) Hydrodynamic diameter, PDI and zeta-potential of

liposomes (blank or siRNA-loaded) were measured by DLS. siRNA entrapment

efficiency was measured by Ribogreen assay. (C) Representative TEM image of

siRNA-loaded liposomes. (D) Hydrodynamic diameter distribution of siRNA-loaded

liposomes. (E) Colloid stability of siRNA-loaded liposomes in PBS. (F) The in vitro

biocompatibility of siRNA-loaded liposomes was evaluated by CCK8 assay. (G)

Histological analysis (H&E) of the lung tissues in mice after pulmonary administration

of saline or liposomes. Images were captured at ×200 magnification. (H) TUNEL

staining of the lung sections from mice following pulmonary administration of saline or

liposomes. Images were captured at ×400 magnification. PDI, polymer dispersity index;

TEM, transmission electron microscope; DSL, dynamic light scattering. The data are

represented as the mean ± SEM.

Supplementary Figure 10. Confocal immunofluorescence image for the

biodistribution of liposomes in epithelial cells after BLM injection. Images were

captured at ×200 magnification.

Supplementary Figure 11. Flow cytometry analysis of the percentage for

macrophages in the lungs originated from Mbd2-C and Mbd2-CKO mice.

Supplementary Figure 12. Single cell sequencing data of IPF lungs.

Supplementary Table 1. Characteristics of subjects for lung samples

Lung samples

COVID-19

(N = 2)

SSc-ILD

(N = 3)

IPF

(N = 8)

Control

(N = 6)

Age (years) 68.00 ± 4.00 57.33 ±

3.333

58.50 ±

3.645

55.17 ± 6.024

BMI 21.64 ±

1.395

23.98 ±

0.547

22.18 ±

1.170

23.40 ±

0.3360

Sex

Female

Male

0 (0.00%)

2(100.00%)

2 (66.67%)

1 (33.33%)

3 (37.50%)

5 (62.50%)

2 (33.33%)

4(66.67%)

FVC

Percent

Predicted

NA

72.87 ±

9.608

65.04 ±

4.565

NA

DLCO NA 52.43 ±

14.88

42.10 ±

4.011

NA

FVC: Forced vital capacity DLCO: diffusion capacity for carbon monoxide