4.1.5. Tissue Preparation for Immunofluorescence and Lung Vascular Morphometry Studies

A separate group of pups were euthanized at one and two weeks of life (*n* = 6/exposure/time-point), and their lungs were inflated and fixed via the trachea with 10% formalin at 25 cm H2O pressure for at least 10 min. Sections of the paraffin-embedded lungs were obtained for immunofluorescence studies and for the analysis of lung vascularization, as described previously [59].

#### 4.1.6. Immunofluorescence Studies

We performed double immunostaining with anti-pERK1/2 and anti-von Willebrand factor (vWF; endothelial specific marker) antibodies to localize ERK1/2 activation in the lung endothelial cells. Fresh frozen lung tissues were incubated with 7.5% normal donkey serum for 1 h to block nonspecific

protein binding, after which they were incubated overnight at 4 ◦C with the following primary antibodies: anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA; 4370, dilution 1:150) and anti-vWF (Abcam, Cambridge, MA, USA; ab11713, dilution 1:50). The primary anti-pERK1/2 and anti-vWF antibodies were detected by incubation with fluorescein-conjugated donkey anti-rabbit (Alexa Fluor 488, dilution 1:200) and donkey anti-sheep (Alexa Fluor 633, dilution 1:200) secondary antibodies, respectively. An indirect TUNEL assay was used to detect apoptosis using the ApopTag Fluorescein In Situ Apoptosis detection kit (MilliporeSigma, St. Louis, MO, USA; S7110), as per the manufacturer's recommendations. The localization of the apoptotic process in the endothelial cells was determined using vWF antibodies, as described above. All the slides were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy. The observers analyzing these slides were masked to the experimental conditions.

#### 4.1.7. Analyses of Pulmonary Vascularization

Pulmonary vessel density was determined in these animals on the basis of the immunofluorescence staining with anti-vWF antibody (Abcam, Cambridge, MA, USA; ab11713, dilution 1:50), which is an endothelial-specific marker [60]. At least 10 counts from 10 random non-overlapping fields (original magnification, 200×) were performed for each animal (*n* = 6/exposure/time-point). The observers performing these measurements were masked to the slide identity.

#### *4.2. In Vitro Experiments*

#### 4.2.1. Cell Culture

The human pulmonary artery endothelial cells (HPAECs) derived from the lungs of human fetus (26 weeks gestational age) were obtained from ScienCell research laboratories (San Diego, CA, USA; 3100). HPAECs were grown in 95% air and 5% CO2 at 37 ◦C in specific endothelial cell medium, according to the manufacturer's protocol. Briefly, the cells were grown in fibronectin-coated plates containing basal endothelial cell medium supplemented with fetal bovine serum, antibiotics, and an endothelial cell growth supplement in a humidifier containing 5% CO2 at 37 ◦C. When the cell culture reached >90% confluence, the cells were subcultured with a split ratio of 1:3. Cells between passages 5–7 were used for all our experiments.

#### 4.2.2. Hyperoxia Exposure

Hyperoxia experiments were conducted in a plexiglass, sealed chamber into which a mixture of 95% O2 and 5% CO2 was circulated continuously. The chamber was placed in a Forma Scientific water-jacketed incubator at 37 ◦C. Once the O2 level inside the chamber reached 95%, the cells were placed inside the chamber for up to 48 h [61]. The cells were harvested at 24 h and 48 h of exposure to determine the effects of hyperoxia on ERK1/2 activation.

#### 4.2.3. Cell Treatment

HPAECs were treated with either 0.01% *v*/*v* dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA; 276855) or the ERK1/2 inhibitor PD98059 (Sigma-Aldrich, St. Louis, MO, USA; P215) at varying concentrations up to 30 μM. The cells were then harvested to determine the effects of PD98059 on ERK1/2 activation, cell proliferation, migration, tubule formation, and expression of cell cycle regulatory proteins.

#### 4.2.4. Western Blot Assays

The cells were grown in complete medium on six-well plates to 70–80% confluence, after which they were exposed to normoxia (95% air and 5% CO2) or hyperoxia (95% O2 and 5% CO2) for up to 48 h. In a separate set of experiments, the cells grown on six-well plates were treated with DMSO or 30 μM PD98059 for 24 h. Following these treatments, whole-cell protein extracts were obtained by using the radio immunoprecipitation assay lysis buffer system (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-24948) and subjected to western blotting with the following antibodies: anti-β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-47778, dilution 1:1000), anti-cyclin A (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-751, dilution 1:1000), anti-cyclin D1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-8396, dilution 1:250), anti-cyclin-dependent kinase (Cdk) 4 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-23896, dilution 1:250), anti-p27 Kip 1 (Abcam, Cambridge, MA, USA ; ab32034, dilution 1:1000), anti-total ERK1/2 (Cell Signaling, Danvers, MA, USA; 4695, dilution 1:1000), anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA; 9106, dilution 1:1000). The immunoreactive bands were detected and quantified as described in the "in vivo experiments" section.

#### 4.2.5. Cell Proliferation Assay

Cell proliferation was determined by a colorimetric assay based on the ability of viable cells to reduce the tetrazolium salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (American Type Culture Collection, Manassas, VA, USA; ATCC 30-1010K) to formazan. HPAECs were grown in 96-well microplates overnight at a density of 5 × <sup>10</sup><sup>4</sup> cells per well in 100 <sup>μ</sup>L of complete medium, followed by an additional period of growth for 24 h in basal medium containing 0.5% fetal bovine serum (FBS). The cells were then treated with varying concentrations of PD98059 and grown under reduced serum conditions for up to 48 h, after which cell proliferation was assessed by the MTT reduction assay, as outlined in the MTT Assay protocol (American Type Culture Collection, Manassas, VA, USA). Briefly, at the end of the experiments, 10 μL of MTT reagent was added to each well, and the cells were incubated in a humidifier containing 5% CO2 at 37 ◦C for 2 h, at the end of which precipitates were visible in all wells. Following the incubation, 100 μL of detergent was added to each well, and the cells were incubated at room temperature in the dark for additional 2 h; the absorbance was measured at 570 nm. The absorbance readings are directly proportional to the number of cells.

#### 4.2.6. Scratch Assay

A scratch assay was used to quantify cell migration [62]. HPAECs were grown in complete medium in six-well plates to 70–80% confluence, followed by a period of growth for 24 h in basal medium containing 0.5% fetal bovine serum. To mitigate the effects of cell proliferation on wound closure, the cells were pre-treated with mitomycin (10 μg/mL) (MilliporeSigma, St. Louis, MO, USA; M4287) for 2 h. The cells were then scratched with a 200 μL pipette tip before they were treated with PD98059. The wound closure or cell migration area was estimated using Image J software (National Institutes of Health, Bethesda, MD, USA) by comparing the wounded areas at 0 h and 16 h.

#### 4.2.7. Tubule and Mesh Formation Assays

A Matrigel assay was used to determine tubule and mesh formation, as described previously [63,64]. HPAECs were grown in 96-well microplates at a density of 2 × <sup>10</sup><sup>4</sup> cells per well in 100 <sup>μ</sup>L of basal medium containing 0.5% FBS. The cells were pretreated with 30 μM PD98059 for 30 min and loaded on top of growth factor-reduced Matrigel (Corning, NY, USA; 356230). Following an incubation period of 18 h, tubule and mesh formation were quantified.
