Fig 2: 1,25(OH)2D3 upregulates the expression of Bcl-2 and downregulates the expression of Bax and Caspase-3. (a, b) Immunofluorescence staining and quantitative analysis of Bax (scale bar = 50 µm). (c, d) Immunofluorescence staining and quantitative analysis of Bcl-2 (scale bar = 50 µm). (e, f) Immunofluorescence staining and quantitative analysis of Caspase-3 (scale bar = 25 µm). Immunofluorescence staining was observed by a fluorescence microscope. (g, h) The protein expressions and quantitative analysis of Bcl-2, Bax, and Caspase-3 in the different groups. (i) Quantitative analysis of the mRNA expression levels of Bcl-2, Bax, and Caspase-3. All data are the mean ± SD. *P < 0.05 and **P < 0.01 compared with the control group; #P < 0.05 and ##P < 0.01 compared with the H2O2 group; &P < 0.05 and &&P < 0.01 compared with the 1,25(OH)2D3 group. LY: LY294002 group.

Fig 3: (A) Representative images showing IL-1ß staining. (B) Immunohistochemistry score performed based on staining incidence of IL-1ß. (C) Representative images showing Caspase-3 staining. (D) Immunohistochemistry score performed based on staining incidence of Caspase-3. Arrows show positively stained areas. Results are expressed as the mean ± standard deviation (n = 3). * p < 0.05; ** p < 0.01. Significance according to one-way ANOVA with post hoc tests, performed using GraphPrism 6.01 software.

Fig 4: TLR4 deficiency impaired apoptosis of neutrophils, with pulmonary up-regulation of Bcl-2. (A) Numbers of apoptotic ex vivo eosinophils and neutrophils were determined by flow cytometry. (B) Representative immunohistochemistry of Bcl-2 in the bronchial regions. Original magnification was 400×. (C-E) Protein expressions of Bcl-2, procaspase-3, procaspase-8, cleaved caspase-3 and cleaved caspase-8 in lung homogenates were detected by Western blotting and densitometric analysis was performed (n = 6).WT, wild-type; TLR4, toll-like receptor 4; TDI, toluene diisocyanate; NS, not significant; Bcl-2, B-cell lymphoma 2; BALF, bronchoalveolar lavage fluid; PI, propidium iodide.*P < 0.05; †P < 0.01; ‡P < 0.001.

Fig 5: TSSK4 promotes AT-II loss through cellular apoptosis.A–C Representative lung sections from fibrotic models as in Fig. 1A were subjected to FITC-TUNEL staining and AT II co-immunofluorescence stained with anti-SP-C antibody. Nuclei were detected with DAPI (A); five individual mice of each group with three random fields of (A) performed to analyze the TUNEL-positive ratio in total AT-II cells by ImageJ program. The scatterplot represents the average value of each mouse (B); representative lung sections were co-immunofluorescence stained with anticleaved caspase-3 antibody, anti-GP36 for AT I, anti-SP-C antibody for AT II, and DAPI for nuclei (C). D Representative lungs from lentivirus-mediated interfering TSSK4 expression and bleomycin-induced fibrosis models as in Fig. 2 were subjected to caspase-3 activity analysis. E–G MLE-12 wild-type cells were transiently transfected with shTSSK4 or scramble shRNA (shCtrl) as control. In the TSSK4 knockout cells that MLE-12 stably transfected with shTSSK4, expression vector (TSSK4+) was transfected to mediate TSSK4-persistent expression. Empty vector (Vector+) was used as control. Different cells were stimulated without or with TNF-a (20 ng/ml) for various durations as indicated. Protein levels of TSSK4 and PARP with cleavage part were analyzed by immunoblotting, with a-tubulin as internal control (E); caspase-3 activity in cellular lysis of shTSSK4 and TSSK4+ was analyzed (F); apoptotic cells were detected by Annexin V/propidium iodide (PI) staining and analyzed by flow cytometry (G). Data in (B, D, F, G) are presented as mean ± s.d. *p < 0.01, **p < 0.01, n.s. p > 0.05, as analyzed by two-tailed unpaired Student’s t test. All data represent 2–3 individual experiments with similar results.