Stefanie C Hummler, Min Rong, Shaoyi Chen, Dorothy Hehre, Deepthi Alapati, Shu Wu. Online Data Supplement

Transcription

1 Targeting GSK-3 to Prevent Hyperoxia-induced Lung Injury in Neonatal Rats Stefanie C Hummler, Min Rong, Shaoyi Chen, Dorothy Hehre, Deepthi Alapati, Shu Wu Online Data Supplement

2 Human Lung Specimens Paraffin embedded lung tissue sections prepared from autopsy specimens were obtained from 4 patients with BPD and 3 newborn patients without lung disease. The BPD patients were born between 23 to 28 weeks of gestational age and died between 3 to 9 months of chronological age. Diagnosis of BPD was made based on standardized criteria including oxygen requirement for more than 28 days at 36 weeks postmenstrual age (1). The primary diagnosis at the time of death for all BPD patients was respiratory failure. Two of these patients also had evidence of systemic infection. The non-bpd patients born between 34 to 36 weeks of gestational age were diagnosed with trisomy 18, complex congenital heart disease and anencephaly and died within 2 to 6 h. Expression of GSK-3 and pgsk-3 was assessed by immunostaining with anti-gsk- 3β and anti-phospho-gsk-3β antibodies (brown signals) and manually semiquantified based on the staining intensity from 0 (no staining) to 4 (strongest staining) (16). Bronchoalveolar Lavage and Analysis Rat pups were sedated with Ketamine (40 mg/kg) and Xylazine (3 mg/kg) by i.p. injection. Animals were tracheotomized with a 22-gauge angiocatheter which was secured in place with a suture. Ice cold normal saline (0.5 ml) was instilled into the airway and gently withdrawn for 4 times. The lavage was repeated four times to recover a total volume of 1.5 to 2 ml. The cells were stained with trypan blue and total cell counts were performed with a hemocytometer. Cytospin (Cytospin 2; Shandon, Waltham, MA) slides were prepared from the BAL fluid and were then fixed and stained using Neat Stain Hematology Kit (Polysciences, Inc., Warrington, PA). A total of 300 cells/slide were viewed for differential cell count.

3 Lung Morphometry For mean linear intercept (MLI) quantification, 10 random images were taken with the 20x magnification on each tissue section. The images were viewed under a field of equally spaced horizontal lines and MLI was assessed as the average of total length of lines divided by the total intercepts of alveolar septa from each lung. Secondary septa count was performed on the same images and the average was calculated for each lung. Radial alveolar count (RAC) was analyzed by identifying 10 terminal respiratory bronchioles under the 10x magnification on each H&E stained tissue section. The number of distal air sacs that were transected by a line drawn from a terminal respiratory bronchiole to the nearest pleural surface was calculated and RAC was determined as the average number of distal air sacs from each lung. Immunostaining and Double Immunofluorescence Staining The following primary antibodies were used in immunostaining and immunofluorescence staining: a rabbit anti-vonwillebrand factor (vwf) antibody from Dako (Carpinteria, CA); mouse anti-α-smooth muscle actin (α-sma) and anti- -catenin antibodies from Sigma (Saint Louis, MI); a rat anti-mac3 antibody from BD Biosciences (San Jose, CA); a rabbit anti-ki67 antibody from Abcam (Cambridge, MA); rabbit anti-gsk-3β and anti-phosho-gsk-3β antibodies from Cell Signaling Technology (Boston, MA). Tissue sections were deparaffinized in xylene and rehydrated through graded ethanol into TBS. The sections were incubated with the respective primary antibodies overnight at 4 C. For immunostaining, the lung sections were then incubated with biotinylated secondary antibodies for 1h at room temperature. Cell-bound biotinylated secondary antibodies were detected with streptavidin-biotin-peroxidase complexes and 3,3 - diamidinobenzidine (DAB) substrates (Vector, Burlingame, CA). For immunofluorescence staining, the lung sections were then incubated with AlexaFluor 488 and AlexaFluor594 labeled secondary antibodies (Invitrogen, Carlsbad, CA) for 1h at room temperature. The slides were

4 washed with TBS, counter stained with 49,6-diamidino-2-phenylindole (DAPI; VectorLab, Burlingame, CA) and mounted. Pulmonary Vascular Morphometry To determine vascular density, immunofluorescence staining for vwf, an endothelial marker, was performed. Ten random images were taken under the 20x magnification on each lung section and the average number of vwf stained vessels (<50 μm in diameter) was calculated. Assessment of Pulmonary Vascular Remodeling For medial wall thickness (MWT) quantification, 20 peripheral pulmonary vessels (<50 μm in diameter) on each lung section were viewed on vwf and α-sma stained tissue sections. The external diameters minus internal diameters were calculated and the average was determined for assessing MWT. Muscularized vessels were defined by the presence of smooth muscle cells positively stained with α-sma antibody in 50% or more of the vessel circumference. Twenty vessels on each lung section were viewed and the percentage of muscularized peripheral pulmonary vessels was determined. Proliferation of vascular smooth muscle cells was assessed by double immunefluorescence staining for α-sma and Ki67, a proliferation marker on lung sections. Twenty peripheral pulmonary vessels on each lung section were viewed and the number of Ki67 positive vessels was calculated. Immunofluorescence staining for -catenin and DAPI nuclear counter staining were performed to determine -catenin nuclear translocation, and the percentage of vessels with at least 1 positive -catenin nucleus was calculated. Western Blot Analysis Total protein was extracted from frozen lung tissues with a RIPA buffer according to manufacturer s protocol (Santa Cruz). The protein concentrations were measured by BCA

5 protein assay using a commercial kit from Pierce Biotechnology Inc (Rockford, IL). Total proteins (50 g/sample) were fractionated by SDS-PAGE on 4-12% Tris-glycine precast gradient gels (Invitrogen) and then transferred to nitrocellulose membranes (Amersham, Piscataway, NJ). The membranes were incubated overnight at 4 o C with respective primary antibodies and then incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. Antibody bound proteins were detected using ECL chemiluminescence methodology (Amersham). Membranes were then stripped with 0.2N NaOH and re-incubated with primary antibodies reactive with a normalization protein, -actin. The intensities of protein bands were quantified by Quantity One Imaging Analysis Program (Bio-Rad, Hercules, CA).