Online Supplement ALVEOLAR CELL SENESCENCE IN PATIENTS WITH PULMONARY EMPHYSEMA. Takao Tsuji, Kazutetsu Aoshiba, and Atsushi Nagai

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1 Online Supplement ALVEOLAR CELL SENESCENCE IN PATIENTS WITH PULMONARY EMPHYSEMA Takao Tsuji, Kazutetsu Aoshiba, and Atsushi Nagai

2 MATERIALS AND METHODS Immunohistochemistry Deparaffinized tissue sections were incubated with 3% hydrogen peroxide for 10 minutes at room temperature to inhibit endogenous peroxidase activity before blocking the nonspecific binding sites with 3% bovine serum albumin and 2% normal goat serum. The sections were then incubated for one hour at room temperature with rabbit polyclonal antisurfactant protein-a (SP-A) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or mouse monoclonal anti-cd31 antibody (DAKO Japan, Tokyo, Japan). The primary antibody was reacted with a secondary antibody conjugated with an alkaline-phosphatase-labelled polymer (EnVison; DAKO), and immunoreactants were visualized by an NBT/BCIPbased reaction. After removal of the prior immunoreactants in a glycine-hcl buffer (ph 2.2), the slides were incubated with mouse monoclonal anti-p21 CIP1/WAF1/Sdi1 antibody (Santa Cruz), mouse monoclonal anti-p16 INK4a antibody (Santa Cruz), or mouse monoclonal anti-proliferating cell nuclear antigen (PCNA: Santa Cruz) antibody. The primary antibodies were reacted with a secondary antibody conjugated with a HRPlabeled polymer (EnVison), and immunoreactants were

3 visualized by a DAB-based reaction. Before anti-cd31 and anti-p21 CIP1/WAF1/Sdi1 immunostaining, sections were autoclaved in a 10 mm citrate buffer (ph 6.0) for 15 minutes to expose the antigen epitopes. Before anti-pcna immunostaining, sections were pretreated with 2 N HCl. Replacement of the primary antibodies with the same concentration of nonimmunized IgG did not result in positive staining. For immunofluorescence staining, the primary antibodies were reacted with secondary antibodies conjugated with either Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes, Eugene, OR). FISH for Telomeres Deparaffinized lung tissue sections were microwaved in a citrate buffer, ph 6.0, for 20 minutes and then treated with a pepsin solution for 10 minutes at 42 o C. The sections were rinsed with deionized water followed by a series of 70%, 85%, and 99% ethanol solutions for 1 minute each, and then air-dried. The sections were incubated for 30 minutes at 37 o C in a 0.1 x SSC solution containing 0.1% NP-40 and then for 10 minutes at 90 o C a 0.1 x SSC solution containing 80% formamide. The sections were again rinsed with deionized water followed by a series of 70%, 85%, and

4 99% ethanol solutions for one minute each, and air-dried. A 10 µl volume of a Cy3-labeled telomere-specific peptide nucleic acid solution (DAKO) was applied to each section, and the sections were coverslipped and denatured by incubation for 3 minutes at 80 o C. The slides were then transferred to an incubator, and hybridization for telomeres was performed at 42 o C for 48 hours. Next, the sections were washed three times, 10 minutes each time, at 45 o C in a 0.1 x SSC solution containing 80% formamide, and then three times, 5 minutes each time, at 45 o C in a 0.1 x SSC solution. For double staining, the sections were exposed to 3% bovine serum albumin and 2% normal goat serum for 30 minutes and then incubated with anti-sp-a or anti- CD31 antibody for one hour. The primary antibody was reacted with a secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR) and counterstained with 4,6-diamidino-2-phenylindole (DAPI). The replacement of the Cy3-labeled telomere-specific peptide nucleic acids with the same concentration of control peptide nucleic acids did not result in positive staining. Quantitative Analysis of p16 INK4a and p21 CIP1/WAF1/Sdi1 Expression by Alveolar Cells

5 Ten microscopic fields of each section doublestained for p16 INK4a or p21 CIP1/WAF1/Sdi1 followed by staining for SP-A or CD31 were randomly selected and examined at a magnification of x200 under a light microscope (Olympus BX60; Olympus Optical Co., Ltd., Tokyo, Japan). A single observer (TT) who was not informed of the clinical data examined each field and manually counted the total number of alveolar type II cells positive for SP-A, the total number of alveolar endothelial cells positive for CD31, the number of cells positive for both SP-A and p16 INK4a, the number of cells positive for both SP-A and p21 CIP1/WAF1/Sdi1, the number of cells positive for both CD31 and p16 INK4a, or the number of cells positive for both CD31 and p21 CIP1/WAF1/Sdi1. The percentages of the total number of cells positive for SP-A that were positive for both SP-A and p16 INK4a or positive for both SP-A and p21 CIP1/WAF1/Sdi1, and the percentages of the total number of cells positive for CD31 that were positive for both CD31 and p16 INK4a or positive for both CD31 and p21 CIP1/WAF1/Sdi1 were calculated for each field, and the mean value was calculated for each patient. More than 500 cells were evaluated on each slide.

6 Measurements of the Telomere Length of Alveolar Cells Twenty to thirty fields of each section hybridized for telomeres and immunostained for either SP-A or CD31 were randomly selected and examined under an epifluorescence microscope (Olympus BX60) at a magnification of 1000x. Digitized video images of each field were captured with an Olympus DP50 CCD camera and sent to an Olympus imaging microscopic workstation (CUSL2G40; Olympus) equipped with a computer running Microsoft Windows XP and WinRoof semiautomated image analysis software (Win Roof Version 3.5, Mitani Corporation, Fukui, Japan). The integration time for all images was 50 milliseconds for Cy3 signal capture, 3 milliseconds for DAPI counterstain, and 100 to 200 milliseconds for Alexa Fluor 488-conjugated antibodies. The images for Cy3 signal capture and DAPI counterstaining were segmented on gray-value thresholding, and the sum of pixel intensities for telomeres in a given cell nucleus was normalized to the DAPI signal by comparing the mean ratios of the telomere signal to the DAPI signal. A single observer (TT) who was uninformed of the clinical data analyzed 100 nuclei of cells positive for SP-A and 100

7 nuclei of cells positive for CD31 from each patient and calculated the mean telomere signal intensity in alveolar type II cells and alveolar epithelial cells. Previous studies have demonstrated that telomere signal intensity is linearly correlated with telomere length (11,12). Schmorl reaction Lipofuscin staining by the Schmorl reaction was performed by incubating sections for 5 minutes at room temperature in a solution containing 0.75% ferric chloride and 0.1% potassium ferricyanide (15). Statistical Analysis All data are expressed as the mean ± SEM or the median, as appropriate. Differences were evaluated for significance by an analysis of variance and the Scheffe test for clinical data, and by the Kruskall-Wallis test and Mann- Whitney test for histological data. Correlations were analyzed by the Spearman rank correlation test. A p value of <0.05 was considered statistically significant. All calculations were performed using StatView 1.0 software for Macintosh (Abacus Concepts, Inc). Intraobserver reproducibility was assessed by calculating the coefficient of variation (CV) for three repeated measurements, and the

8 CV for telomere FISH, lipofuscin measurements, and immunohistochemistry was 3%, 5%, and 6%, respectively. Figure E1. Representative results of immunofluorescence staining of lung tissue sections from emphysema patients. Lung tissue sections were double-stained with antibodies against anti-p16 INK4a (red) and anti-sp-a (green) (A), antip21 CIP1/WAF1/Sdi1 (red) and anti-sp-a (green) (B), anti-p16 INK4a (red) and anti-cd31 (green) (C), and anti-p21 CIP1/WAF1/Sdi1 (red) and anti-cd31 (green) (D). Arrows indicate cells that were positive for both p16 INK4a and SP-A (A), cells that were positive for both p21 CIP1/WAF1/Sdi1 were positive for both p16 INK4a and anti-sp-a (B), cells that and CD31 (C), and cells that were positive for both p21 CIP1/WAF1/Sdi1 and CD31 (D). Original magnification, x 1000.