Fig 2: TSSK4 knockdown ameliorates bleomycin-induced lung fibrosis.C57BL/6 mice were intratracheally infected with lentivirus-mediated shTSSK4 or control vectors (1.05 × 1010 infectious units (IFUs) in a volume of 30 µl per animal), and then intratracheally treated with bleomycin (3 mg/kg body weight) or the same amount of saline for a period as indicated. A H&E staining and Masson’s trichrome staining of the representative lungs from fibrosis model mice as indicated. B Quantitative mRNA expression of the fibrotic genes, including Timp1, Col3, MMP12, and a-SMA in the lungs of fibrosis models as (A) was detected through qPCR (n = 5/group). C Hydroxyproline assay of the lungs from different groups as indicated was performed (n = 5/group). D Relative body-weight loss of model mice was detected for a period of 24 days (n = 10/group). E, F IHC staining with anti-TSSK4 and anti-SP-C antibodies in the representative lungs from fibrotic model mice (E); five individual mice of each group with three random fields of (E) performed to analyze the relative signal intensity of TSSK4 and number of AT-II cells in the alveolar area through ImageJ program. The scatterplot represents the average value of each mouse (F). G Immunoblot analysis of TSSK4 and SP-C level in the lungs of different fibrotic groups at different time points as indicated, with GAPDH as internal control. Data in (B, C, D, F) are presented as mean ± s.d. In (B, C, F), **p < 0.01, n.s. p > 0.05, as analyzed by two-tailed unpaired Student’s t test. In (D), **p < 0.01, was analyzed by one-way ANOVA test. All data represent 2–3 individual experiments with similar results.

Fig 3: Loss of CPEB2 reduces pulmonary myofibroblasts (MYFs) during alveologenesis. a Caveolin 1 (Cav1+)-immunostained images from postnatal day 3 (P3) CP2WT and CP2KO lungs with magnified images of selected areas shown in a’. The number of Cav1+ type I alveolar epithelial cells (AECs) was quantified from 5 mice per genotype. b,c Similar to a, pro-surfactant-C–positive (SFTPC+) type II AECs and aSMA+ MYFs counted in magnified images of selected areas shown in b’ and c’ (5 mice per genotype). d The number of aSMA+ MYFs counted in CP2WT and CP2KO lungs (5 mice per group) at denoted ages. Data are mean ± s.e.m., *P < 0.05 and ***P < 0.001, by Student’s t test in a-c and by two-way ANOVA and Bonferroni post-hoc test in d. Scales, 50 µm in a-d and 25 µm in a’-c’

Fig 4: SARS-CoV-2 tropism in human lung xenografts. Sections of SARS-CoV-2-infected lung xenografts harvested at 6 dpi were subjected to multiplex immunofluorescence assay for (A) co-staining of SARS-CoV-2 N protein (N, green), human ACE2 (hACE2, yellow), human CK-19 (hCK-19, red) and human vimentin (hVimentin, magenta), or for (B) co-staining of SARS-CoV-2 N protein (green), human ACE2 (hACE2, yellow), human SP-C (hSP-C, red) and human PDPN (hPDPN, grey). Nuclei were stained with DAPI (blue). White frame was magnified on the right and bottom panels. Solid white arrows indicate the SARS-CoV-2+/hACE2+ cells. Scale bars in the upper left panels in (A) and (B) represent 50 µm, while those in other panels represent 20 µm. Staining was performed on tissue sections from two lung xenografts obtained from two donors, and representative images are shown.

Fig 5: Subcutaneous transplantation of human fetal lung tissues into SCID mice to develop the human lung xenograft mouse model. (A) Scheme of construction of the human lung xenograft mouse model. Small fragments (~10 mm3) of human fetal lung tissues were transplanted subcutaneously (s.c.) into the right flank of SCID mice. After 8 weeks, the lung xenografts can grow and develop mature lung structures. (B) Gross view and (C) histological analysis of the human lung xenograft at 8 weeks post-transplantation. (D) Immunohistochemistry staining of lung marker expression in the human lung xenograft. Sections of human lung xenografts at 8 weeks post-transplantation were immunostained for human CK-19 (hCK-19), human vimentin (hVimentin), human PDPN (hPDPN), human SP-C (hSP-C), human CC10 (hCC10) to visualize the lung epithelium and mesenchyme, type I and type II pneumocytes, and bronchiolar Clara cells, respectively. Tissue sections were also stained for human ACE2 (hACE2) expression. The lower panels represent higher magnification images of the areas outlined by the dotted lines in the upper panels. Scale bars in the left and right panels in (C) represent 1 mm and 200 µm, respectively. Scale bars for 100 × and 400 × magnification in (D) represent 200 µm and 50 µm, respectively. Staining was performed on tissue sections from 4 lung xenografts obtained from two donors, and representative images are shown.