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- Supplementary methods -
Immunofluorescence Staining
Cell Culture and Irradiation
Cell Culture and Bleomycin Treatment
Guinea Pig Exposure to Cigarette Smoke
Lung Processing, Morphometric Analysis, and Immunofluorescence Staining - Figure S1 - Correlations between the numbers of γH2AX foci and the numbers of phosphorylated 53BP1 foci per cell in type II cells (A) and between the numbers of H2AX foci and numbers of phosphorylated ATM substrate foci per cell in type II cells (B). Circles = asymptomatic smokers; triangles = asymptomatic smokers; squares = COPD patients.
- Figure S2 - X-irradiation of lung microvascular cells induces DSBs, apoptosis, cell senescence, NFκB phosphorylation, and IL-6 production. Normal human lung microvascular cells were cultured and irradiated with a 10 Gy X-ray dose. Cells were fixed in 3% paraformaldehyde 2 hours later for immunostaining for γH2AX, phosphorylated 53BP1, and active caspase-3, or 48 hours later for immunostaining for p16 and phosphorylated NFκB. Cell culture supernatants were harvested 48 hours after irradiation, and IL-6 the concentration was measured by ELISA. (A) Representative images of immunofluorescence stained X-irradiated cells (IR) and unirradiated cells (Cont). Positive immunosignals are seen in red fluorescence. Cell nuclei were stained with DAPI. (B-F) Quantitative analyses of the results of immunofluorescence staining. N = 4 in each experiment. (G) Results of ELISA measurements of the amount of IL-6 secreted by X-ray-irradiated cells and unirradiated cells. N = 8 in each experiment.
- Figure S3 - Treatment of A549 cells with bleomycin induces DSBs, apoptosis, cell senescence, NFκB phosphorylation, and IL-6 production. A549 cells were treated with 50 μg/ml of bleomycin and fixed in 3% paraformaldehyde 24 hours later in preparation for immunostaining for γH2AX, phosphorylated 53BP1, and active caspase-3, or 48 hours later for immunostaining for p21 and phosphorylated NFB. Cell culture supernatants were harvested after 48 hours, and the IL-6 concentration was measured by ELISA. (A) Representative images of immunofluorescence-stained bleomycin-treated cells (Bleo) and untreated cells (Cont). Positive immunosignals for γH2AX, phosphorylated 53BP1, active caspase-3, and phosphorylated NFκB are seen in the form of red fluorescence, and for p21 in the form of green fluorescence. Cell nuclei were stained with DAPI. (B-F) Quantitative analyses of the results of immunofluorescence staining. N = 4 in each experiment. (G) Results of ELISA measurements of the amount of IL-6 secreted by bleomycin-treated cells and untreated cells. N = 8 in each experiment.
- Figure S4 - DSBs occurred in the lungs of guinea pigs with cigarette smoke-induced emphysema. (A) Representative photomicrographs of lung tissue sections stained with hematoxylin and eosin (upper panels) and stained with anti-γH2AX antibody (lower panels). Inset is an enlarged photomicrograph of γH2AX-positive cells. (B) Quantitative analyses of airspace size (Lm: mean linear intercepts) and the number of γH2AX foci in the alveolar wall cells. CS = lungs of guinea pigs exposed to cigarette smoke for 10 weeks (n = 5); Air = lungs of sham-exposed guinea pigs (n = 8). Data are expressed as means � SEM.
- Supplementary methods -
Immunofluorescence Staining