Fstl1

Equal amounts of protein extracted from lung tissue, HPASMCs or plasma (1 μL) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (0.45 μm, Millipore, USA). After blocked by 5% non-fat milk for at least 1 h at room temperature, the membrane was incubated in primary antibodies of FSTL1 (1:500, Santa Cruz, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000, Cell Signalling Technology, USA), p-ERK, t-ERK, p-AMPK, t-AMPK, p-Smad 1/5/8, t-Smad 1/5/8, p-p38, t-p38, p-JNK and t-JNK (1:200, Cell Signalling Technology, USA) overnight at 4 °C. Then the membrane was washed in PBST (0.1% Tween 20-PBS, Sigma-Aldrich, USA) 3 × 10 min followed by incubation in IRDye800-conjugated secondary antibody (1:10000, Odyssey LI-COR, USA) for 1 h. After washed again in PBST 3 × 10 min, the membrane was visualized by the LI-COR Odyssey imaging system. The protein signal was quantified by Image J and relative protein level was normalized to that of GAPDH as house-keeping protein or to their respective total expressions.

Serial sections in thickness of 4 μm were cut through the paraffin-embedded left lung lobe and stained with HE (Beijing Dingguo Changsheng Biotechnology, China). Anti-α-SMA (1:200, Sigma-Aldrich, USA) and FSTL1 (1:200, Santa Cruz, USA) were visualized by Alexa Fluor 594-labelled goat anti-mouse IgG and Fluorescein-conjugated rabbit anti-goat IgG (ZsBio, China), respectively, with nuclei mounted by 4′,6-diamidino-2-phenylindole (DAPI, ZsBio, China). Blood vessels were screened with a microscope digital camera (Nikon, Japan) and analyzed by NIS-Elements system (Nikon, Japan). Vascular remodelling was evaluated by MT% and numbers of completely muscularized arterioles52 (link)53 (link). Briefly, MT% was expressed as a percentage of ((external diameter - internal diameter)/external diameter). Arterioles exhibiting more than 75% of circumference positive for α-SMA were identified as completely muscularized arteries and their numbers were totaled in every 10 high-power (×400) fields. Transversely cut arterioles were included for measurement, with the exclusion of obliquely cut ones and pulmonary veins.