Fig 2: Effects of Areg deficiency on the tubule formation ability of fetal mouse lung endothelial-like cells. Matrigel assay was performed to quantify the tubule formation ability using fetal mouse lung endothelial-like cells transfected with control or Areg siRNA and exposed to normoxia (21% O2 and 5% CO2, n = 5/group) or hyperoxia (70% O2 and 5% CO2, n = 5/group). (A–D) Representative photographs showing tubule formation of cells transfected with control (A,C) or Areg (B,D) siRNA and exposed to normoxia (A,B) or hyperoxia (C,D). (E) Quantification of tubule formation. Scale bar = 100 µm. Data are expressed as mean ± SD. Analysis of variance was used for the statistical analyses. ns = not significant. Significant differences between exposures are indicated by *, p < 0.05, and ****, p < 0.0001.

Fig 3: Hyperoxia (HO) exposure increases Areg mRNA in neonatal murine lungs. (A) Experimental design for Figure 1 and Figure 2. O2—oxygen, P—postnatal day, and RT-PCR—real-time polymerase chain reaction. Whole-lung mRNA was extracted from neonatal murine lungs after 14 d of 21% O2 (normoxia; n = 3) or 70% O2 (hyperoxia; n = 4) exposure and subjected to RT-PCR analysis to quantify Areg RNA expression (B). Data are expressed as mean ± SD. T-test was used for the statistical analyses. Significant differences between exposures are indicated by *, p < 0.05.

Fig 4: Overview of the results. O2—oxygen, BPD—bronchopulmonary dysplasia, ECs—endothelial cells, Areg—amphiregulin, and ERK—extracellular signal-regulated kinase.

Fig 5: Areg treatment efficiently increases Areg protein expression in the cell culture supernatant of fetal mouse lung endothelial-like cells. Areg protein expression was quantified by ELISA in the cell culture supernatant of fetal mouse lung endothelial-like cells treated with the vehicle, phosphate-buffered saline (PBS), or up to 100 ng/mL of recombinant mouse Areg and exposed to normoxia (21% O2 and 5% CO2, n = 3/group) or hyperoxia (70% O2 and 5% CO2, n = 3/group). Values are presented as mean ± SD. Analysis of variance was used for the statistical analyses. Significant differences between exposures are indicated by **, p < 0.01 and ****, p < 0.0001.