α sma

The section (5 µm) from lung tissues were washed with PBS and separately co-incubated with HSP110 (Affinity) and YAP (Proteintech), HSP110 (Affinity) and alpha smooth muscle actin (α-SMA) (Affinity), YAP (Proteintech) and α-SMA (Abclonal), proliferating cell nuclear antigen (PCNA) (Abclonal) and α-SMA (Affinity), and Beclin1 (Abclonal) and α-SMA (Affinity) antibodies overnight at 4 °C. After washing, the sections were subject to second antibodies Cy3-labeled goat anti-rabbit IgG (Beyotime; Invitrogen, USA) and FITC-labeled goat anti-mouse IgG or anti-rabbit IgG (Beyotime; Abcam, UK) incubation at room temperature for 1.5 h. For cell immunofluorescence staining, fixed glass coverslips with cells were incubated with primary antibodies α-SMA (Affinity), Beclin1 (Abclonal) and YAP (Proteintech) overnight at 4 °C. Then the cells were subsequently incubated with fluorescent secondary antibodies Cy3-labeled goat anti-mouse IgG or anti-rabbit IgG (Beyotime) at room temperature for 1 h. The sections and coverslips then were incubated with DAPI (Aladdin, China) for counterstain and pictures were taken with fluorescence microscope (Olympus).

The liver tissue was immediately fixed in a 10% formaldehyde solution and gradient ethanol for dehydration treatment, then embedded in paraffin to make 5 μm thick tissue sections. Before pathological staining, the slices were placed in an oven at 60 ℃ for 2 h, then dewaxed with xylene and rehydrated with gradient ethanol. Tissue sections were then stained with H&E, Sirius Red (Cat.MM1004, MaoKang biotechnology Co., Ltd, Shanghai, China), Masson (Solarbio, Peking, China), and were performed to observe the structural changes and collagen fiber deposition of the liver tissues in each group by an optical microscope.
For immunohistochemical staining, the tissue sections were dewaxed and rehydrated with gradient ethanol. After antigen repair with hydrogen peroxide and sodium citrate, the tissue sections were sealed with 5% BSA at room temperature for 1 h. Tissue sections were incubated overnight at 4 ℃ with the following primary antibodies from Affinity Biosciences (Cincinnati, OH, USA): α-SMA (Cat. AF1032), Collagen I (Cat. AF7001) at a dilution of 1:200. After washing 3 times by PBS, the anti-rabbit IgG (Cat. ZB-2301, Zhongshan-golden bridge, China) secondary antibodies was incubated at 37 ℃ for 1 h. DAB color solution was added for color development, and the nuclei were stained with hematoxylin. Tissue sections were observed with light microscopy and photographed.

PTL (> 99%) was provided by Shangdeyaoyuan Co. (Tianjin, China). DEX sodium phosphate (> 98.5%) was purchased from Meilun Biological Technology Co. (Dalian, China), and BLM sulfate (> 91%) was obtained from Meilun Biological Technology Co. (Dalian, China). The NF-κB, Snail, β-actin, GAPDH, E-cadherin, vimentin, MMP1, α-SMA and Col-1 antibodies were purchased from Affinity Biosciences Co. (Beijing, China). The mouse TNF-α, mouse IL-4, mouse TGF-β1, and mouse interferon gamma ELISA Kits were purchased from Meilian Biological Technology Co. (Shanghai, China). Chlorine ammonia T (> 97.08%) and p-dimethylaminobenzaldehyde (> 97.08%) were obtained from (> 99.71%). Reverse-4-hydroxy-l-proline (> 99.4%) was purchased from Bailingwei Technology Co. (Beijing, China). Perchloric acid (> 70%) was obtained from Jingchun Biological Technology Co. (Shanghai, China).