Fig 1: DAMPs are involved in lung I/R injury-triggered autophagy in alveolar macrophages of minipigs. (a) ELISA of HMGB1 and HSP60 production in the BALF of the sham group or minipigs subjected to lung I/R as indicated (n=3). (b) Immunohistochemical staining of HMGB1 and HSP60 in left lung tissues of the sham group or minipigs subjected to lung I/R as indicated. (c) Immunoblotting analysis of LC3, BECN1, SQSTM1 and ß-actin (as a loading control) in lysates of alveolar macrophages treated with rpHMGB1 (1 µg/ml) or rpHSP60 (1 µg/ml) for the indicated periods. (d) Immunofluorescence analysis of LC3 in alveolar macrophages stimulated with rpHMGB1 (1 µg/ml) or rpHSP60 (1 µg/ml) for 3 h. Original magnification, × 630. Quantification of cells with autophagosomes is also shown. (e) Immunoblotting analysis of LC3, SQSTM1 and ß-actin (as a loading control) in lysates of alveolar macrophages with or without treatment with E64d (15 µg/ml), Pepstatin A (15 µg/ml) or Bafilomycin A1 (30 nM) before stimulation by rpHMGB1 (1 µg/ml) or rpHSP60 (1 µg/ml) for 3 h. Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1 and SQSTM1) to ß-actin in the same lane. The relative band intensities of LC3-II/ß-actin were calculated from three independent experiments and shown as mean±S.E.M. Data are mean±S.E.M. (a) or representative (b–e), of three individual experiments. *P<0.05, **P<0.01, ***P<0.001