Fig. 6.

The effect of propranolol on the tubulogenic properties of the combined IH-derived EC, PER, and TC depends on IH-TC. Isolated IH cells are cultured and amplified before they were labeled with three different fluorescent dyes: EC in red, PER in blue, and TC in green. EC, PER, and TC were seeded on microslide wells covered with a thin layer of Matrigel. Tube formation was then imaged 4 h later and analyzed using Wimtube software from Wimasis. (A and B) Substitution of IH-TC by foreskin-TC impaired the responses of the three-cell-type model of IH to 3 and 10 µM of propranolol. (A) Representative fluorescent images with illustrative analysis image. (B) Total tubes, average quantification of three wells per condition (n = 3), representative results from three different patients (n = 3). (C) Western blotting evaluation of AQP1 protein level in IH-TC in control medium or after 2 h with 3 μM propranolol. Protein intensity was normalized to β-actin. The mean relative protein amount was calculated based on three different experiments (n = 3). (D) AQP1 in IH-TC was down-regulated using shAQP1. Then, shCT- and shAQP1-transduced TCs were used in our in vitro three-cell-type–based tubulogenesis assay. Data are expressed as mean ± SEM. Statistical analysis was performed with one-way ANOVA with uncorrected Fisher’s LSD. *P < 0.05, **P < 0.005, and ****P < 0.00005; ns, not significant.