Alexafluor 633 tagged phalloidin

Human lung autopsy tissues from infants that died with BPD and infants that died at similar postnatal ages from non-pulmonary causes (control) were obtained from Dr. Gloria Pryhuber and collected as part of the BRINDL repository and Lung Map consortium [28 (link)]. Study inclusion/exlusion criteria, IRB approval and parental consent has been previously described [29 (link)]. Lung tissue sections were obtained from 16 infants, 8 infants that died with BPD and 8 infants that died of other non-pulmonary causes, (n = 8 in each group) and the clinical descriptions and co-morbidities of the infants from whom these samples were collected have been previously published [29 (link)]. F-actin content in tissue was estimated by using Alexafluor-633 tagged phalloidin (Invitrogen, Eugene, Oregon) and DAPI (Invitrogen, Carlsbad, CA) for the nucleus. Images were acquired in a blinded fashion using a confocal microscope (LSM510, Carl Zeiss, Jena, Germany) with 40X objective. Four photomicrographs were obtained from each tissue section assessed independently and averaged for each individual thus creating an n = 8 for each diagnosis. Identical confocal settings were applied to acquire all images across conditions. F-actin content/cell was quantified using NIH Image J analysis.

BM-MSCs were seeded on cover slips coated with type 1P collagen (1 × 10⁴ cells/cm²) and cultured in the culture medium for 24 h. Then, the cells were starved with serum-free DMEM supplemented with 1% l-glutamine and 1% P/S for another 18 h. The cells were then treated with PC3 CM or CON CM for 24 h, and subsequently fixed with 4% PFA. After fixation, immunocytochemistry was performed according to standard protocols [32 (link)]. The following primary antibodies were used: Anti-N-cadherin (1:30; Invitrogen), Anti-N-cadherin (1:100; Takara, Shiga, Japan), anti-β-catenin (1:100; Bethyl Laboratories, Montgomery, TX, USA), and anti-α-catenin (1:100; BD Biosciences, San Jose, CA, USA). Actin was stained with Alexa Fluor 633-tagged phalloidin (1:1000; Invitrogen). Nuclei were stained using DAPI for 10 min. Images were taken using a Zeiss LSM 700 confocal microscope (Carl Zeiss) at a 400× magnification. The number and the length of N-cadherin-positive structures at the borders between BM-MSCs and the width of N-cadherin-positive borders between BM-MSCs were measured (Figure S1) using Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA).