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Figure 1. MiR-449 affects actin network remodelling and multiciliogenesis in HAECs.(a–c) Effect of a treatment by control antagomiR (CTR-Neg), anti-miR-449a/b (Antago-449) and miR-449::Notch1 protector (PO-Notch1) on differentiating HAECs. (a) Staining for F-actin (a1,5,9), ezrin (a2,6,10) and acetylated tubulin (a3,7,11), at LC stage. (b) The histogram indicates the average percentage of MCCs (in magenta) and apical ezrin-positive (in green) cell number relative to control (means±s.d. from nine and three donors for MCC and ezrin quantifications, respectively. ***P<0.001, **P<0.01; Student's t-test). (c) Immunostaining of focal adhesions protein Paxillin (in green), F-actin (in red) and nuclei (in blue) in A549 epithelial cells transfected for 72 h with control miRNA (miR-Neg), miR-449a or miR-Neg plus 10 μM DAPT. (d) Ratio of focal adhesion number per cell, normalized to control (n=5 fields in three independent experiments; ***P<0.001; Student's t-test). (e) Effect of miR-449 overexpression and DAPT (10 μM) on ERM phosphorylation in proliferating HAECs. Phosphorylated protein levels were normalized with non-phosphorylated ERM and with an antibody against HSP60 as a loading control. Normalized fold changes are indicated on the corresponding bands. Experiments were representative of three donors. (f) Effect of PO-Notch1 and DAPT (10 μM) in differentiating HAECs at LC stage on miR-449 expression, normalized with RNU44. (g) Real-time RT–PCR of HES1 transcripts in control, DAPT (10 μM, 48 h)-treated or miR-449-overexpressing proliferating HAECs. Transcript levels of HES1 were normalized against UBC transcript as an internal control. (f,g) Data represent the mean and s.d. of three independent experiments (***P<0.001, **P<0.01; Student's t-test). Image published in: Chevalier B et al. (2015) Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |