Fig 2: Cathepsin expression and gross phenotype of Ctsh-deficient mice.(A) Western blots for Ctsh detection in lungs and liver of Ctsh+/+ and Ctsh -/- mice. (B) Detection of “acidic” aminopeptidase activity at pH 6.0 in lungs, livers and kidneys of f Ctsh+/+ and Ctsh -/- mice (n = 3). (C) Observed and expected genotype frequencies of litters from Ctsh +/- x Ctsh +/- matings. (D) Weight gain of female littermates from heterozygous matings (n = 5 per genotype). (E) mRNA expression of cathepsin C (Ctsc) and cathepsin E (Ctse) measured by quantitative ‘real-time’ RT-PCR in lungs of Ctsh+/+ and Ctsh -/- mice (n = 5 per group). (F) Cathepsin C (Ctsc) and cathepsin E (Ctse) detected by Western blotting in Ctsh+/+ and Ctsh -/- lung lysates.

Fig 3: Cathepsin H function in production of lung surfactant proteins.(A) mRNA expression of surfactant proteins A1, B, and C measured by quantitative ‘real-time’ RT-PCR in lungs of Ctsh+/+ and Ctsh -/- mice (n = 5 per group). (B) Detection of surfactant protein B (SP-B) in lung tissue lysates (C) Western blot detection of SP-B in broncho-alveolar lavage (BAL) of Ctsh+/+ and Ctsh -/- mice of 2 genetic backgrounds (129P2/OlaHsd and C57BL/6N). The lysosomal membrane associated protein 2a (Lamp 2a) is present at the limiting membrane of lamellar bodies [27], [28] and serves as loading control independent of the surfactant proteins. (D–F) Surface activity of BAL fluid measured by pulsating bubble surfactometry (n = 6–10).

Fig 4: Cathepsin H expression in the lung.(A) Expression of the lacZ reporter (blue staining) in Ctsh +/- mice as indication for Ctsh transcription in distinct alveolar cells, i.e. type II pneumocytes (arrows heads), and (B) in small airway bronchioles (indicated by *). (C–D) Histology and absent immunohistochemical detection of Ctsh in Ctsh -/- lungs. (E–F) Immunohistochemical detection of Ctsh in lungs of wild-type mice confirms the expression of Ctsh in distinct pneumocytes and bronchiolar epithelium (indicated by *). Scale bars are 50 µm.