Hydroxyproline Assay Kit (Cat.No.MAK008-1KT, Sigma-Aldrich, St. Louis, MO), PowerUp SYBR-Green Master Mix kit (Cat. No. A25742) and a High Capacity cDNA Reverse Transcription kit (Cat. No. 4368814) were obtained from Applied Biosystems (Foster City, CA). NuPAGE 10% Bis-Tris gel (Cat.No.NP0301BOX, Invitrogen, Carlsbad, CA). Bleomycin (Cat.No. HY-17565, MedChemExpress, LLC, NJ), The antibodies used in this study were showed in Table S1.
A mouse model of bleomycin-induced pulmonary fibrosis
H19-/- (H19 ΔExon1-5) mice were generated by CRISPR/Cas9-mediated genome engineering in C57BL/6J mice as our previously described (20). The animal procedures were approved by the Shanghai Jiao tong University School of Medicine affiliated Xin Hua hospital Animal Care and Use Committee (XHEC-F-2020-008). The mice (about 8 weeks old) were divided into four groups: wild type (Wt) sham (n=8-12), H19-/- sham (n=6-10), wild type (Wt) treated with bleomycin (BLM) (n=8-12) and H19-/- treated with bleomycin (BLM) (n=8-12). For bleomycin administration, mice were anaesthetized with 2% isoflurane, and then instilled intratracheally with bleomycin (3.5 mg/kg) in 100 μL PBS, as previously described (25, 26). On day 28, the lung was collected for RNA, protein, collagen content analyses and histological analysis. The degree of fibrosis was quantitated using an Ashcroft score in a blinded manner according to the described method (27).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from left lung of mice using the RNeasy kit (Qiagen, Hilden, Germany) according to the protocol of the manufacture and 2 mg of total RNA was used to synthesize the 1st cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The real-time PCR reactions were performed on the ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA) using PowerUp SYBR-Green Master Mix kit (Applied Biosystems, Foster City, CA). All samples were assayed in triplicate, and data were normalized to endogenous control Hprt1. Relative RNA expression levels were calculated using the ΔΔCt method. The primers are listed in Table S2.
A total 30 mg left lung tissues was homogenized in 300 μL RIPA buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Servicebio, Wuhan, China). After determining the protein concentration, the equal amounts of protein were separated on NuPAGE 10% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred onto polyvinylidene diﬂuoride (PVDF) membranes. After blocking in 5% nonfat milk at room temperature for 60 min, membranes were incubated with the primary antibodies overnight at 4°C.The membranes were washed three times for 30 min with TBST (containing 0.1% Tween-20), and then incubated with secondary antibodies. After ﬁnal washes with TBST, the signals were detected using ECL chemiluminescence reagent Kit (Pierce, Rockford, IL, USA). The primary antibodies performed in this study as showed in Table S1.
Histology and immunofluorescence (IF)
The right lung tissues were immediately fixed in 10% neutral buffered formalin for 24 h and and go through dehydration, clearing and paraffin embedding. Sections were mounted on positively charged slides after cutting at 4 mm thick, baked at 65 °C for 1h and then stored at room temperature (RT) for later use. Fibrosis was performed using mason’s trichrome (Genmed Scientifics, Wilmington, DE, USA) and Sirius red stain (Servicebio, Wuhan, China) following the protocols of manufactures. For immunofluorescence (IF) assay, the slides were incubated with xylol and descending concentrations of ethanol. Endogenous peroxidases were blocked by using 0.3% H2O2 for 10 min at RT. After antigen retrieval, blocking was performed using 5% bovine serum albumin for 30 min at RT. The antibodies used here were listed on the Table S1.
The amount of collagen in the lung tissues was determined by A Hydroxyproline Assay Kit according to the manufacturer's protocol (Cat.No.MAK008-1KT, Sigma-Aldrich, St. Louis, MO). Briefly, about 10 mg lung tissues were homogenized in 100 μl water with 100 μl concentrated hydrochloric acid (HCl, 12 M) and hydrolyzed at 120°C for 3 hours. Transfer 20 μl of supernatant at 60°C until completely desiccated. Chloramine T/Oxidation Buffer Mixture was added at room temperature for 5 minutes, followed by the addition of Diluted DMAB Reagent and incubation at 60°C for 90 minutes. Measure the absorbance of samples and standards at 560 nm, and hydroxyproline content was expressed as μg per mg lung tissue.
All data were expressed as mean ± SD (standard deviation). For comparisons of different groups, statistical significance was determined by Student's t-test or ANOVA analysis. P-value less than 0.05 was considered statistically significant.