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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: Eur Respir J. 2021 Apr 15;57(4):2000652. doi: 10.1183/13993003.00652-2020

Figure 4. A BH3 mimetic ABT-263 attenuates the expression of MAVS and lung fibrosis.

Figure 4.

(a-e) The indicated BH3 mimetics with 100 μM amounts, respectively, were treated for 3 h. (a) MLE-12 cells were treated with the indicated BH3 mimetics after 10 mU/ml bleomycin treatment for 3 days. The expression of MAVS was evaluated by Western blot analysis. (b) The mitochondrial fractions of MLE-12 cells treated with the indicated BH3 mimetics after 10 mU/ml bleomycin treatment for 3 days, respectively. The expression of MAVS and Oxidative phosphorylation (OXPHOS) complexes were evaluated by Western blot analysis. Voltage-dependent anionic channel (VDAC) protein was evaluated as loading controls of mitochondria. (c) The mouse embryonic fibroblasts (MEF) cells were treated with the indicated BH3 mimetics. The expression of MAVS was evaluated by Western blot analysis. (d) Primary peritoneal macrophages (PM) were isolated from the peritoneal cavity of wild type mice. The cells were treated with 10 and 100 μM ABT-263. The expression of MAVS was evaluated by Western blot analysis. (e) Primary murine lung fibroblasts (MLF) were isolated from wild type murine lungs. The cells were treated with ABT-263. The expression of MAVS was evaluated by Western blot analysis. (f) The Scheme of the experimental approach, and evaluation results of the total lung collagen contents from wild type murine lungs are presented. The mice were administered with bleomycin (+) and treated after day 8 with ABT-263 (40 mg/kg, every 2 d, i.p.), and sacrificed at day 21. Each dot indicates the individual mouse used for the experiment. (n=5 per each of both control groups, n=23 per bleomycin only treatment group, n=20 per bleomycin+ABT-263 treatment group, respectively). (g) Western blot evaluations for p-SAMD2, SMAD2/3, Fibronectin and a-SMA proteins from whole lung tissue lysates, respectively, at day 14 after bleomycin administration are presented. (n=5 per each of the groups). (h) After stimulation with 20 ng/ml TGF-β1 for 24 h, MEF, MLF and human normal lung fibroblasts (NHLF) were treated with 100 μM ABT-263 for 3 h. Then, the expression of p-SMAD2, SMAD2/3, respectively, were evaluated by Western blot analysis. For panels (a), (c-e) and (g-h), β-Actin was used as a loading control. Data are the mean ± SEM. Statistical significance was calculated using the 2-way ANOVA with Tukey’s multiple comparisons test (f) ****, P < 0.0001.