Mouse model of IUI and Serpine1 siRNA treatment

Pregnant C57BL/6 mice at embryonic day 12.5 (E12.5) were randomly divided into either control or LPS groups. Mice in the LPS group were intraperitoneally injected with 45 μg/kg LPS (Escherichia coli serotype 055: B5, Sigma-Aldrich), while the control group received an equal volume of saline. Lung tissues were collected from neonatal mice on postnatal day 1 (P1), P7, and P21, and stored at −80 °C before analysis. For Serpine1 small-interfering RNA (siRNA) treatment, P3 neonatal mice with IUI were administered Serpine1 siRNA (5`-CCAACAAGAGCCAAUCACATT-3`) or negative control siRNA (RiboBio) via intranasal delivery at a dose of 0.25 nmol/g/day for 4 days. Lung tissues were then collected on P7.

Cell culture and treatments

MLE-12, A549, BEAS-2B and human epithelial kidney 293 T (HEK293T) cell lines were purchased from ATCC. MLE-12, A549 and BEAS-2B cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco). HEK293T cells were grown in DMEM supplemented with 10% FBS. Cells were treated with 5-Aza-CdR (0.125, 0.25, 0.5 μM; Sigma), TSA (10, 25, 50 nM; Sigma), or TNF-α (10 ng/mL, 100 ng/mL; Abcam) for 24 h.

Plasmid construction and transfection

Serpine1 open-reading frame was amplified with the cDNA from C57BL/6 murine lung tissue and cloned into the pCI-neo vector (Promega). The JASPAR database was used to predict the potential transcription factor binding sites on the Serpine1 promoter. Rela, Hif1a, and Rest were cloned into the pCMV-SPORT6 vector. Three shRNAs were designed to repress Hdac2 expression. All constructs were confirmed via sequencing. All vectors were transfected into cells using Lipofectamine 2000 (Invitrogen). Cells transfected with empty vector or scrambled shRNA vector were used as controls.

Immunohistochemistry and immunofluorescence

Paraffin slices were dewaxed using xylene I and II (10 min each time), hydrated with gradient alcohol (100, 95, and 75%, 5 min each), and washed with PBS (three times, 5 min each). Antigen retrieval was performed in 1 × Tris–EDTA buffer at 100 ℃ for 20 min. After cooling to room temperature, the slices were incubated with 3% hydrogen peroxide solution for 10 min at room temperature and washed with PBS. Then, the slices were blocked with 3% BSA (Yeasen) for 60 min at 37 ℃ and probed with the following the primary antibodies: rabbit anti-CD31 (1:2000; Abcam), rabbit anti-SFTPC (1:2000; Abcam), rabbit anti-Tet1 (1:500; Abcam), and rabbit anti-Hdac2 (1:500; Abcam), in the wet box overnight at 4 ℃. For immunohistochemistry, the slices were washed with PBS (three times, 5 min each) and incubated with the HRP-linked goat anti-rabbit secondary antibody (1:500; Yeasen) for 60 min at 37 ℃. After PBS washes, the slices were stained with diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, cleared, sealed, observed under an inverted microscope, and photographed. For immunofluorescence, the slices were probed with Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:1000; Invitrogen) for 60 min at 37 ℃. After three PBS washes (5 min each), the cells were stained with 4',6-diamidino-2-phenylindole (DAPI, Beyotime) for 10 min to visualize nuclei. Five fields under the microscope (× 200 or × 400) were randomly selected for evaluation using the ImageJ software.

RT-qPCR

The total RNA was extracted using the RNeasy Mini Kit (Qiagen). The OD 260/280 nm ratio was measured using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using TaKaRa PrimeScript 1st strand cDNA Synthesis kit (TaKaRa). Messenger RNA levels were quantified with a QuantStudio (TM) 7 Flex System (Applied Biosystems) using SYBR® Green PCR Master Mix (AG BIO). The data were analysed using the 2−ΔΔCt method.

Western blot

Tissues or cells were lysed on ice with precooled lysis buffer for 30 min and centrifuged at 12 000 rpm for 10 min at 4 ℃. The supernatant was transferred to centrifuge tubes, and 5 × SDS loading buffer was added. Samples were denatured for 10 min at 99 ℃ and subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Merck). The membrane was blocked with 5% BSA for 60 min at room temperature and incubated with the primary antibodies against Serpine1 (1:1000; Abcam), Nr4a1 (1:1000, Abcam), β-actin (1:1500, Yeasen), Gapdh (1:1500, Yeasen), and transferrin (1:1000, Beyotime) overnight at 4 ℃. After washing three times with 1 × TBST, the membrane was probed with the HRP-linked goat anti-rabbit secondary antibody (1:1000, Yeasen) for 60 min at room temperature and washed with 1 × TBST buffer. Proteins were visualized using electrogenerated chemiluminescence reagents (Epizyme) and detected with an ImageQuant LAS 4000 chemiluminescent Image Analyzer (General Electric). The relative densitometric density of the target band to the internal reference band was analysed using the ImageJ software.

Cell proliferation assay

Cell proliferation staining was performed using a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime). Briefly, cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 48 h. Subsequently, cells were incubated with EdU for 2 h at 37 ℃ and washed three times with PBS. Then, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS and permeated with 0.3% Triton X-100 for another 15 min at room temperature. After three PBS washes, the cells were incubated with the Click Reaction Mixture for 30 min at room temperature with protection from light and then incubated with Hoechst 33,342 for 10 min. Five fields under the microscope (× 200) were randomly selected for the evaluation.

Senescence-associated β-galactosidase activity

Cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 48 h. The SA-β-Gal staining was performed using a Senescence β-galactosidase Staining kit (Beyotime), and the cells were examined under a light microscope (Olympus BX51). Cellular senescence was expressed as the percentage of SA-β-Gal-positive cells to the total cell number.

Cell apoptosis analysis

Cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 48 h. The apoptotic cells were detected using FITC Annexin V Apoptosis Detection Kit (Dojindo) followed by flow cytometry analysis.

Cell cycle assay

Cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 48 h. Then, cells were harvested and fixed overnight using 75% ethanol. After incubation with 50 μg/mL propidium iodide dye (Yeasen) and 100 μg/mL RNase A (Yeasen) for 30 min, flow cytometry (BD Biosciences) was performed to quantify the percentage of cells in each cell cycle phase (G0/G1, S, and G2/M).

ROS content measurement

Cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 24 h. Then, intracellular ROS content was measured using a ROS assay kit (Yeasen). The intracellular ROS content were expressed as fluorescence intensity. Excitation/emission wavelengths were 490/585 nm.

Assessment of mitochondrial membrane potential

Cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 24 h. Then, the membrane potential of isolated mitochondria was measured by JC-1 staining (Beyotime). Mitochondria containing JC-1 aggregates were detected in the red (excitation 525 nm, emission 590 nm) and those containing JC-1 monomers were detected in the green channels (excitation 490 nm, emission 530 nm). The ratio of red to green fluorescence signal intensity indicated the mitochondrial membrane potential.

Cell immunofluorescence

Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS (5 min each time), permeabilized with 0.25% Triton X-100 for 10 min at room temperature, washed with PBS (three times, 5 min each), and blocked with 5% BSA for 60 min at 37 ℃. The cells were then incubated with the following primary antibodies: rabbit anti-Tet1 (1:500, Abcam) and rabbit anti-H2AX (1:400, Abcam), overnight at 4 ℃. Cells were washed with PBS and probed with Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:1000; Invitrogen) for 60 min at 37 ℃. After three PBS washes (5 min each), the nuclei were stained with DAPI for 10 min. Five microscope fields (× 200) were selected randomly for the evaluation. The mean fluorescence intensity was analysed using the ImageJ software.

DNA methylation analysis

MethPrime program was used to predict the potential methylation sites of Serpine1 and design the primers (Serpine1-BSP-F: 5′-GGAATTTGATATTTTTTAGTTTTTATATG-3′ and Serpine1-BSP-R: 5′-CCTACTTCTACCTCCTAAATACTCC-3′) for bisulfite sequencing PCR (BSP). Bisulfite conversion and purification were performed using the EpiTect bisulfite Kit (Qiagen) per the manufacturer’s instructions. Bisulfite-converted DNA was amplified with TaKaRa EpiTaq HS (TaKaRa), and the 139-bp PCR product was purified with the TaKaRa MiniBEST agarose Gel DNA Extraction Kit (TaKaRa). Amplified fragments were inserted into the pMD™18-T vector (TaKaRa) and transformed into Escherichia coli Trans5α. Plasmid DNA from individual clones was isolated and subjected to Sanger sequencing using M13F primers. The methylation status of each CpG locus was determined by sequence comparison using the QUMA software.

Luciferase assay

The 2000 bp 5′UTR (untranslated region) fragment of Serpine1 was amplified from genomic DNA of C57BL/6 mice using the primers Serpine1-NheI-F: 5'-AATGCTAGCCACCATGCAGATGTCTTCAGC-3' and Serpine1-EcoRI-R: 5'-ATTGAATTCTCAAGGCTCCATCACTTGGCCCAT-3'. The amplified PCR products were cloned into the PGL3-basic vector (Promega) to generate the PGL3-Serpine1 construct. The PGL3-Serpine1 vector was incubated with the CpG methyltransferase M.SssI (NEB) for 4 h at 37 °C to methylate all CpG residues, followed by purification of vectors with the Cycle Pure Kit (Omega). Complete methylation was verified via BstUI digestion (NEB). Vectors were co-transfected into 80% confluent MLE-12 or HEK293T cells. After 48 h of incubation, a Dual-Luciferase Reporter Assay System (Promega) was used to measure the relative luciferase activities.

ChIP

ChIP was performed using a SimpleChIP Enzymatic chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s instructions. Treated or mock-treated (0.5 μM 5-Aza-CdR for 24 h) MLE-12 cells were crosslinked with 37% formaldehyde. Chromatin was fragmented via partial digestion with micrococcal nuclease before sonication. The lysate was incubated at 4 °C overnight with anti-Rela and anti-Hdac2 rabbit monoclonal antibodies, followed by incubation with Protein G Magnetic Beads for 2 h at 4 °C. After reversal of protein-DNA crosslinks at 65 °C for 2 h, the DNA was purified using DNA purification spin columns, and immunoprecipitated DNA was analysed via RT-qPCR using a pair of primers (Serpine1-Chip-F:5'-CTGCTGCCTCCCTTTATACCA-3' and Serpine1-Chip-R: 5'-CATGCCCTTTCACACGTACAC-3'). A distant region amplified by primers (NC-F: 5'-CCCCGTGACCTGGATTTGAT-3' and NC-R: 5'-TACTATTGTGGGGGTCGGGA-3') was used as control. Fold enrichment method was used to normalize ChIP-qPCR data.

DNA pull-down

The Serpine1 promoter was amplified from genomic DNA of C57BL/6 mice using a pair of 5'-biotinylated primers (Serpine1-biotin-F: 5'-TGGTATCTGTTTACTGGAAATGGAAAACA-3' and Serpine1-biotin-R: 5'-GCTCCGTTCCCTGGCTGA-3'), corresponding to -2000 bp upstream the translation initiation site of Serpine1. A distant region amplified by primers (NC-biotin-F: 5'- GGCACATACTCTGCCACTGA-3' and NC-biotin-R: 5'-TACTATTGTGGGGGTCGGGA-3') was used as the control. Nuclear protein from mouse lung tissues was extracted using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Next, 50 μg of Dynabeads® M-280 Streptavidin (Invitrogen) was incubated with 1 μg of the promoter DNA for 1 h at 4 °C with rotation. After that, beads-DNA was washed three times with one volume of binding buffer (5 mM Tris–HCl, pH 7.5, 0.5 mM EDTA, and 1.0 M NaCl), and then resuspended in one volume of incubation buffer (50 mM Tris, 1 mM EDTA, 100 mM KCI, pH 7.0, 5% glycerol, and 0.1% Triton X-100). Nuclear proteins (100 μL) were added and incubated with beads overnight with rotating at 4 °C, followed by three washes before liquid chromatography-mass spectroscopy (LC-MS) and western blot analyses. The LC–MS was performed by Lumingbio Co., Ltd., (Shanghai, China).

Statistical analysis

Data are presented as mean ± SEM and analysed using GraphPad Prism version 6.0 software. Statistical significance was determined using Student’s t-test or one-way ANOVA. P values < 0.05 were considered statistically significant. All the experiments were performed at least in three independent repeats.