Fig 1: Effect of ex vivo treatment with Pirfenidone and Nintedanib on lung epithelial cell marker in fibrotic 3D-LTCs. a-c Fibrotic 3D-LTCs were cultured for 48 h in the presence of anti-fibrotic drugs Nintedanib (1 µM) and Pirfenidone (500 µM). a proSP-C expression was assessed by Western blot. ß-Actin was used as loading control. Quantification of proSP-C Western blot, n = 6. Data was normalized to ß-Actin. b, c SP-C and WISP1 secretion of 3D-LTCs was determined by ELISA. n = 4–7. Significance was assessed using Wilcoxon matched pairs test. Significance: *p < 0.05
Fig 2: Ex vivo treatment with Nintedanib stimulates alveolar epithelial marker expression in the human 3D-LTCs model of early pulmonary fibrosis. a Schematic of treatment with fibrotic cocktail (FC) or control cocktail (CC) and Nintedanib (Nint) and downstream analysis. 3D-LTCs were generated and treated with FC or CC for 48 h before FC or CC treatment was replenished and Nintedanib or control treatment was added. Treatment was stopped after 120 h and downstream experiments were performed. b Representative Immunofluorescence of punches treated with CC or FC for 120 h and stained for Fibronectin. Scale bars represent 1 mm. c-f Punches were treated with CC/FC and Nintedanib (1 µM) as indicated in (a). c Metabolic activity of punches 120 h after treatment with CC/FC and co-treatment with Nintedanib. N = 3. Significance was assessed by two-way ANOVA followed by Sidak’s multiple comparisons test. d Gene expression analysis by qPCR of epithelial cell marker SFTPC, NKX2.1, CDH-1, ZO-1. Log fold change is presented as mean ± SEM, N = 3. Means were compared to respective DMSO control using one-sample t-tests in comparison to a hypothetical value of 0. e Representative Immunofluorescence of punches treated with FC and Nintedanib for proSP-C. Scale bars represent 140um. f SP-C secretion of punches 120 h after treatment with CC/FC and co-treatment with Nintedanib was measured by ELISA. Values shown are normalized to CC treatment. Significance was assessed using Wilcoxon matched pairs test. N = 6. Significance: *p < 0.05
Fig 3: Effect of in vitro treatment with Pirfenidone and Nintedanib on primary mouse (pm)ATII cells. a, b At day 14 after Bleomycin instillation, mice were sacrificed and control (PBS) and fibrotic (Bleo) pmATII cells were harvested. The pmATII cells were cultured in the presence of Nintedanib (1 µM) and Pirfenidone (500 µM) for 48 h. a Gene expression analysis by qPCR of fibrotic marker Fn1 in pmATII cells. ?Cp relative to Hprt is presented as mean ± SEM, n = 3. Means were compared using repeated-measures one-way ANOVA followed by Newmann-Keuls post test. b Gene expression analysis by qPCR of epithelial cell markers Sftpc, Nkx2.1, T1a, Hopx. ?Cp relative to Hprt is presented as mean ± SEM, n = 3. Means were compared using repeated-measures one-way ANOVA followed by Newmann-Keuls post test. Significance: *p < 0.05, **p < 0.01, ***p < 0.001 (DMSO vs Pirfenidone/Nintedanib). Significance: #p < 0.05, ##p < 0.01, ###p < 0.001 (PBS vs Bleo)
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