Cell culture and treatment

Human fetal lung IMR-90 fibroblast cells (derived from a 16-week-old female Caucasian fetus) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were cultured in 90% minimum essential medium (MEM) with 2 mM l-glutamine and Earle’s balanced salt solution (BSS) adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (Corning, Corning, NY, USA), and 10% fetal bovine serum (FBS) under 5% CO2 and 95% relative humidity at 37 °C. Cells were exposed to 10, 30, and 50 μg/ml LPS (Escherichia coli O111:B4, Sigma-Aldrich, St. Louis, MO, USA) and control medium (0 μg/ml) for 24 h.

Fetal lung ex vivo culture and treatment

Pregnant ICR mice were obtained from BioLASCO Taiwan (Taipei, Taiwan) and euthanized at E11.5 (at the pseudoglandular stage). Embryos was collected from the mice followed by lung dissection under a dissecting microscope. All lungs were cultured on Transwell® membranes (Corning) with Biggers, Gwatkin, and Judah (BGJb) medium (Gibco, Grand Island, NY, USA) containing 0.1% FBS, l-ascorbic acid, and primocin (InvivoGen, San Diego, CA, USA). Fetal lungs were exposed to 0, 10, 30, and 50 μg/ml LPS under 5% CO2 and 95% relative humidity at 37 °C for 3 days. Medium was collected and replaced everyday. The lung morphology was investigated every 24 h using a Leadview 2000AIO Digital Camera (Taipei, Taiwan). The surface area of the fetal lungs and the number of buds were calculated by ImageJ software (vers. 1.53, National Institutes of Health (NIH), Bethesda, MD, USA) after being normalized to the first day of the experiment.

Lactate dehydrogenase (LDH)

Supernatants were collected from cells and fetal lung ex vivo culture for an LDH cytotoxicity assay (Donjido Molecular Technology, Rockville, MD, USA). Details of the experimental procedures were in accordance with the manufacturer’s instructions.

Enzyme-linked immunosorbent assay

IL-6, IL-8 and chemokine (C-X-C motif) ligand 1(CXCL1) levels in supernatants collected from cells and fetal lung culture were examined using enzyme-linked immunosorbent assay (ELISA) kits (ThermoFisher Scientific, Waltham, MA, USA and R&D Systems, Minneapolis, MN, USA). Details of the experimental procedures were in accordance with the manufacturer’s instructions.

Western blot analysis

Protein from cells and fetal lungs was collected with lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Samples were electrophoresed in 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with non-fat dried milk diluted in Tris-buffered saline/Tween-20 (TBST) for 1 h. Samples were incubated with primary antibodies including mouse anti-YAP (1:1000; Proteintech, Rosemont, IL, USA), rabbit anti-p-YAP (1:1000; Abcam, Cambridge, UK), rabbit anti-TAZ (1:1000; Cell Signaling, Danvers, MA, USA), rabbit anti-p-TAZ (1:1000; Cell signaling), rabbit anti-FGF10 (1:1000; Abcam), rabbit anti-FGFR2 (1:1000; Abcam), rabbit anti-SOX2 (1:1000; Cell Signaling), rabbit anti-SOX9 (1:1000; Cell Signaling), rabbit anti-SIRT1 (1:1000; Cell Signaling), rabbit anti p-SIRT1 (1:1000; Signalway Antibody, Greenbelt, MD, USA), and mouse anti-β-actin (1:5000; Proteintech) overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, protein bands were detected with the ChemiDoc™ MP Imaging system (Bio-Rad, Hercules, CA, USA) and quantified by Image-Pro software (vers. 4, Media Cybernetics, Rockville, MD, USA). All data were normalized to the control.

Immunofluorescence (IF)

Paraffin-embedded fetal lung tissue sections were placed in an oven at 60 °C and rehydrated before staining. Antigen retrieval was done by undergoing heating with citrate buffer (pH 6.0). Bovine serum albumin (BSA, Bionova Scientific, Fremont, CA, USA) with 0.25% Triton X-100 was used for cell permeabilization and 5% BSA was used for blocking at room temperature, followed by incubation with a primary antibody, mouse conjugated fibronectin (1:250; Santa Cruz Biotechnology, Dallas, Tx, USA) for 1.5 h and another primary unconjugated antibody consisting of mouse anti-YAP (1:400; Proteintech), rabbit anti-p-YAP (1:400; Abcam), rabbit anti-TAZ (1:500; Cell Signaling), rabbit anti-p-TAZ (1:500; Cell Signaling), rabbit anti-FGF10 (1:250; BOSTER BIO, Pleasanton, CA, USA), rabbit anti-SOX2 (1:400; Cell Signaling), rabbit anti-SOX9 (1:200; ABGENT, San Diego, CA, USA), rabbit anti-SIRT1 (1:400; Cell Signaling), and rabbit anti p-SIRT1 (1:200; Cell Signaling). A fluorophore-conjugated secondary antibody against the primary antibody was used, and then the sample was covered with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Abcam). Samples were imaged by confocal fluorescence microscopy (TCS SP5, Leica, Wetzlar, Germany) equipped with a camera and imaging software (SPOT Imaging, Sterling-Heights, MI, USA) at 400× magnification. The co-expression mean intensities of YAP, p-YAP, TAZ, p-TAZ, FGF10, SOX2, SOX9, SIRT1, and p-SIRT1 that were fibronectin positive (fibronectin+; for identifying fibroblasts) in five different regions were quantified by ImageJ software (NIH) as previously reported (Shihan et al. 2021).

Immunocytochemistry (ICC)

IMR-90 cells were cultured and then fixed by 2% formaldehyde in PBS for 15 min at room temperature before staining. 0.5% Triton X-100 was used for permeabilization and 5% BSA in PBS was used for blocking at room temperature, followed by incubation with a primary antibody including mouse anti-YAP (1:400; Proteintech), rabbit anti-p-YAP (1:400; Affinity, Melbourne, Victoria, Australia), rabbit anti-TAZ (1:400; Cell Signaling) and rabbit anti-p-TAZ (1:400; Cell Signaling) overnight at 4 °C. A fluorophore-conjugated secondary antibody against the primary antibody was used, and then the sample was covered with mounting medium containing DAPI. Samples were imaged by confocal fluorescence microscopy equipped with a camera and imaging software as the above mentioned at 200× magnification.

RNA-sequencing

Total RNA was collected from fetal lung tissues in Trizol® reagent (Ambion, Life Technologies, Carlsbad, NY, USA) after exposure to 50 μg/ml LPS and control medium. The purity and quantification were checked using a SimpliNano™-Biochrom Spectrophotometer (Biochrom, Holliston, MA, USA). A Qsep 100 DNA/RNA Analyzer (BiOptic, New Taipei, Taiwan) was used to monitor RNA degradation and integrity. Sequencing libraries were generated using a KAPA messenger (m)RNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland) following the manufacturer’s instructions. The original data were obtained through the Illumina NOVAseq 6000 platform. Clean data were obtained by evaluating the parameters including low-quality reads, adaptor contamination, and base qualities. A gene ontology (GO) pathway enrichment analysis of differentially expressed genes (DEGs) was performed using clusterProfiler (vers. 4.4.0). A gene set enrichment analysis (GSEA) was used to identify enriched biological functions and activated pathways from a molecular signature database (MSigDB). A dotplot was created by Rstudio 10.14.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD). Continuous variables were examined by a one-way analysis of variance (ANOVA) with Tukey’s post-hoc or an unpaired t-test. Statistical analyses were performed using GraphPad Prism 7 (San Diego, CA, USA). A p value of < 0.05 was considered statistically significant.