Disrupting the miR‐34a/Pdgfra interaction restores myofibroblast abundance and limits hyperoxic damage to the developing alveolar architecture in mouse.

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Disrupting the miR‐34a/Pdgfra interaction restores myofibroblast abundance and limits hyperoxic damage to the developing alveolar architecture in mouse lungs Disrupting the miR‐34a/Pdgfra interaction restores myofibroblast abundance and limits hyperoxic damage to the developing alveolar architecture in mouse lungs AGeneration of two target site blocker (TSB) locked nucleic acid sequences: TSB1 and TSB2 (in blue), for the disruption of the miR‐34a/Pdgfra interaction, indicating binding sites in the Pdgfra 3′‐UTR, Kit 3′‐UTR, and the Sirt1 3′‐UTR (in black), alongside the miR‐34a sequence (in red). The miR‐34a seed sequence, and the seed‐sequence binding site in the target mRNA 3′‐UTR are indicated in bold, and brown, respectively.BEvaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR‐34a (MIM34a) mimics, and probing for PDGFRα and c‐Kit as TSB‐dependent and TSB‐independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and c‐Kit were all probed on the same membrane; hence, a single βactin immunoblot is presented. Data are representative of three experiments.CEvaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR‐34a (MIM34a) mimics, and probing for PDGFRα and SIRT1 as TSB‐dependent and TSB‐independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and SIRT1 were all probed on the same membrane; hence, a single βactin immunoblot is presented. Data are representative of three experiments.DQualitative analysis of lung structure in Richardson‐stained plastic‐embedded lung sections from wild‐type mouse pups at post‐natal day (P)14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O2) and aberrant (85% O2) alveolarization (scale bar, 50 μm). Data are representative of three or more experiments.EQuantification of total number of alveoli by design‐based stereology in wild‐type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).FQuantification of mean septal thickness by design‐based stereology in wild‐type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).GQuantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild‐type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).HQuantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild‐type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).Data information: Data represent mean ± SD. P values were calculated by unpaired Student's t‐test. Source data are available online for this figure. Jordi Ruiz‐Camp et al. EMBO Mol Med. 2019;emmm.201809448 © as stated in the article, figure or figure legend