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. 2023 Aug 31;14:4956. doi: 10.1038/s41467-023-40617-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Heatmap visualization of mRNA expression of SASP factors evaluated by qRT-PCR using normal AOs from Sftpc^CreERT2; Rosa26^mTmG mice and p53-cKO AOs from Sftpc^CreERT2; Trp53^flox/flox; Rosa26^mTmG mice. AOs were treated with 100 μM BLM for 24 h, and harvested on culture day 9. Data were obtained from three independent mice. *P < 0.05, **P < 0.01, ***P < 0.001 compared between BLM-treated normal AOs and BLM-treated p53-cKO AOs (unpaired, two-tailed Student’s t test). b Representative images of lung sections for in situ hybridization and quantification using Sftpc^CreERT2; Rosa26^mTmG mice on days 0 and 7 after BLM injection in vivo. Yellow triangles indicate mRNA-dot-positive GFP⁺ AT2-lineage cells. Data represent the mean ± SEM of results obtained from three independent mice. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired, two-tailed Student’s t test). (scale bar: 100 μm). c FACS panels with quantification of mean fluorescence intensity (MFI) for integrin αVβ6 using GFP⁺ AT2-lineage cells (days 0 and 7, in vivo). Data represent the mean ± SEM of results obtained from three independent mice. ***P < 0.001 compared to day 0 (unpaired, two-tailed Student’s t test). d Experimental set-up, representative images of co-cultured AOs and myofibroblasts (scale bar: 1 mm), and quantification of the relative DsRed⁺ area around the AO-containing gel using normal AOs treated with BLM (100 μM, 24 h) and subsequently with a neutralizing antibody against integrin αVβ6 (100 μg mL⁻¹, 72 h). Data represent the mean ± SEM of results obtained from three independent mice. *P < 0.05 (unpaired, two-tailed Student’s t test). e Diagram of the experiments and comparison of mRNA expression levels by qRT-PCR for primary lung fibroblasts. Lung fibroblasts were isolated from wild-type mice (upper panel) or tamoxifen-treated transgenic mice (lower panel): Pdgfra^CreERT2; Tgfbr2^flox/flox or Pdgfra^CreERT2; Tgfbr2^flox/+. These fibroblasts were treated with culture supernatants from normal AOs that were pre-treated with BLM (100 μM, 24 h) and then cultured for the next 72 h. Data represent the mean ± SEM of results obtained from three independent mice. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Bonferroni correction).