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. 2024 Jan 2;15:87. doi: 10.1038/s41467-023-44421-6

Fig. 5. Soluble Plet1 drives AEC expansion and promotes alveolar epithelial barrier function in ex vivo models.

Fig. 5

a Representative image of BALO co-cultured with BMDM2 for 21D and treated with anti-Plet1 mAb or isotype control. Scale bar represents 50 µm. b Quantification of BALO numbers; single data points represent the mean of organoid numbers per each well (n = 5) (****p < 0.0001). c Mean diameter (µm) of organoids (n = 40–87) per each well, n = 5 well/group, after 7D in culture; single data points represent mean diameter of organoids per each well (****p < 0.0001, ***p = 0.0006). d average numbers of alveoli per BALO after 21D in culture (control) or co-culture with BMDM2 and anti-Plet1 mAb or isotype control (alveoli were counted from n = 3 BALO per group) (*p = 0.0252). e Predicted kinase activity (5 top phosphorylated, red, and de-phosphorylated kinases, green; shown within phylogenetic kinome tree) in AEC treated with 20 ng/ml rPlet-1 for 12 h. f Suggested kinase signaling pathway activated by Plet1. The figure was created with BioRender. g Assessment of Ki67 staining in murine primary AEC culture treated with rPlet1 (20 ng/ml) and/or MEK inhibitor U0126 (10 µM) (n = 3) (*p = 0.0065, **p = 0.004, ***p = 0.0006). h Quantification of tight junction gene expression (Tjp-1, Cldn1, Ocln) by qPCR in murine AEC IAV-infected and treated with rPlet1 (20 ng/ml) (n = 3) (Tjp-1 ***p = 0.0004, ****p < 0.0001; Cldn1 *p = 0.0282, **p = 0.0236; Ocln ***p = 0.0003, ****p < 0.0001). i Representative image of ZO-1 localization in AEC monolayers, IAV-infected and/or treated with rPlet1. Scale bar represents 50 µm. j Time course of transepithelial resistance (TER) in murine (n = 3) and human AEC (n = 4) monolayers on transwells, IAV-infected and/or treated with rPlet1 (*p < 0.0001 for the comparison between AECs+A/PR8 and AECs+A/PR8+rPlet1). k Quantification of tight junction gene expression (Tjp-1, Cldn1, Ocln) by qPCR in murine AEC IAV-infected and treated with rPlet (20 ng/ml) with/without Src activator (10 µM). Results are shown as fold change over IAV-infected AEC without rPlet1 (n = 4) (Tjp-1 ****p < 0.0001; Cldn1 ****p < 0.0001; Ocln **p = 0.0024). Data are representative of three independent experiments showing mean ± SEM calculated using one-way ANOVA and Tukey’s post-hoc tests. j represents a single experiment performed with 3 (mouse) or 4 (human) different biological independent samples showing mean ± SEM and results were analyzed by two-way ANOVA and Tukey’s post-hoc test. BMDM bone marrow-derived macrophage, AEC alveolar epithelial cell, BALO bronchoalveolar lung organoid, TER transepithelial resistance, TR-AM tissue-resident alveolar macrophage, D day, inh inhibitor. Source data are provided as a Source Data file.