Figure 5.

Dethamexasome (DXM) rescues differentiation defects of congenital diaphragmatic hernia (CDH) basal stem cells (BSCs) in air–liquid interface (ALI) cultures, whereas Poly(I:C) stimulation of control BSCs induces differentiation defects similar to those of CDH BSCs. (A) Schematic of the workflow. DXM (10 μM) was added during 1 week of cell expansion and the first week during ALI differentiation of CDH BSCs. (B) Representative fluorescence images of antibody staining for epithelial cell markers in Day 21 ALI cultures of CDH BSCs (n = 5 lines) with and without DXM treatment. Nuclei were stained by 4′,6-diamidino-2-phenylindole. (C) The relative abundance of each labeled cell type was quantified for each condition in triplicates. Each dot represents one CDH BSC line. (D) Representative Western blot for phosphorylated Ser536 in the p65 subunit (phosphorylated nuclear factor kappa B) in CDH BSCs with and without DXM treatment (n = 2) for 1 week during cell culture. β-actin was a loading control. (E) Schematic of the workflow. Poly(I:C) (10 μM) was added for the first week during ALI differentiation of control BSCs. (F) Representative fluorescence images of antibody staining for epithelial cell markers in Day 21 ALI cultures of control BSCs with and without Poly(I:C) treatment. (G) The relative abundance of each labeled cell type was quantified for each condition in triplicates. Each dot represents one control BSC line. ALI culture was performed in triplicates, and more than 6,000 cells were counted using stained sections of fixed ALI cultures for each BSC line. Bar graphs show mean ± SEM. *P < 0.05 and **P < 0.01 by Mann-Whitney U test. Scale bar, 25 μm.