A 5-point semi-quantitative severity-based scoring system was used
to assess the degree of apoptosis: 0 = normal lung parenchyma; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100% of examined tissue. Selleckchem GSK 3 inhibitor Quantification of murine Y chromosome in lung tissue was achieved by quantitative real-time polymerase chain reaction (PCR). Briefly, DNA was purified in a 600 μl solution of 0.2% sodium dodecyl sulfate (SDS)/proteinase K (300 μg/ml), extracted with an equal volume of phenol/chloroform/isoamyl alcohol, and centrifuged for 15 min at 14,000 rpm. The aqueous phase was transferred to a new tube. DNA was precipitated with 2 volumes of ethanol 100% and centrifuged for 15 min at 14,000 rpm. DNA was resuspended and quantified in a nanodrop spectrophotometer. 5 ng of DNA was used in a real-time PCR reaction with SYBR Green detection kit run in 7000-sequence detection system thermocycler according to manufacturer instructions (Applied Biosystems, Foster City, CA). The following PCR primers were used: forward 5′-TCA TCG GAG GGC TAA AGT G-3′; and reverse 5′-CAA CCT TCT GCA GTG GGA C-3′. Primers sequences
were defined using primer3 software based on Mus musculus sex-determining region of Chr Y (Sry) gene, gene bank accession number: NM_011564 (National Institutes of Health, NIH, Bethesda, USA). These primers amplify an 88 bp product. The relative amount of total DNA was Venetoclax ic50 calculated as a ratio (2-ΔCt) of Sry and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers for
GAPDH – Forward: many 5′-CCA CCA ACT GCT TAG CCC-3′ and reverse: 5′-GAC ACC TAC AAA GAA GGG TCC A-3′, 145 bp. In order to evaluate the mechanisms related to lung remodeling, quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR) was performed to measure the expression of transforming growth factor (TGF)-β, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and caspase-3 genes. Central slices of left lungs were cut, collected in cryotubes, quick-frozen by immersion in liquid nitrogen, and stored at −80 °C. Total RNA was extracted from the frozen tissues, using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. RNA concentration was measured by spectrophotometry in Nanodrop® ND-1000. First-strand cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA). PCR primers for target gene were purchased (Invitrogen, Carlsbad, CA).