Fig. 3.

TNFα and IL1α are required for single lumen maintenance. (A) Diagram showing the three different experimental set-ups for studying the role of pro-inflammatory cytokines in maintenance of single central lumen. In set-up 1, epithelial 3D cultures were grown in BIE supplemented with dexamethasone or hydrocortisone (BIE+DEX or BIE+HC) for 6 days. After 6 days, TNFα or IL1α were added to the culture and the percentages of multiple-lumened and single-lumened glands were scored after 6 additional days. In set-up 2, epithelial 3D cultures were grown in BIE for 6 days. After 6 initial days, medium was supplemented with HC or DEX and glands were cultured for 6 additional days. In set-up 3, epithelial 3D cultures were grown in BIE+DEX or BIE+HC for 6 days. At this point, medium was replaced by BIE medium with or without glucocorticoids and cultures grown for 9 additional days. (B) Reversible switch of phenotypes from multiple lumen to one single lumen. Representative images (left) and quantification (right) of single-versus multiple-lumened glands at day 6 and at the end of the first experimental set-up (day 12). (C) Representative images (top) and quantifications (bottom) of single-versus multiple-lumened glands at day 6 and at the end of the second experimental set-up (day 12). (D) Glucocorticoid removal is sufficient to restore single-lumened glands and expression of pro-inflammatory cytokines. Left: Quantification of single-versus multiple-lumened glands at the end of the third experimental set up (day 15). Real-time PCR analysis of TNFα (middle) and IL1α (right) expression at the indicated days of treatment. Results are expressed as relative mRNA levels. (E) Representative images of time-lapse experiments corresponding to experimental set-up 1 (top) and experimental set-up 2 (bottom). Scale bars: 50 μm. *P≤0.05.