Fig. 1.

Expression of a clinical Sftpc mutation challenges AEC2 proteostasis and activates the UPR. (A) Schematic of Sftpc^C185Gneo founder line with LoxP-flanked inhibitory neomycin cassette and post-TMX induced Cre recombination to the active Sftpc^C185G. (B) (Top) Western blotting of AEC2 cell lysate 7 d post-TMX for SP-C shows primary translation product (*) in Sftpc^WT and Sftpc^C185G samples, an absence of SP-C in the Sftpc^C185Gneo founder line, and a failure of Sftpc^C185G SP-C to undergo proteolytic cleavage to intermediate isoforms (}). (Bottom) Western blotting of BALF large-aggregate surfactant fractions for mature SP-C (mSP-C) shows absence of mSP-C in alveolar compartment of Sftpc^C185G mice (n = 2 per genotype). (C) Representative immunostaining of lung section for SP-C shows punctae appearance in Sftpc^WT, an absence of SP-C in the Sftpc^C185Gneo founder line, or a reticular pattern to the Sftpc^C185G SP-C (60× magnification, scale bars = 20 μM). (D) Western blotting analysis for BIP, ATF4, and CHOP in MLE12 cells ± 500 nm tunicamycin (TM) and Sftpc^WT and Sftpc^C185G AEC2 lysates 7 d post-TMX. (E) Ratio of spliced to unspliced Xbp1 as assessed by qRT-PCR of Sftpc^WT and Sftpc^C185G AEC2s. (F) Volcano plot showing the differences in gene expression in Sftpc^WT versus Sftpc^C185G AEC2s (red = >1.5 fold change, adj P value < 0.05). Genes annotated to the proteasome are indicated and show enrichment in Sftpc^C185G AEC2s. (G) Kyoto Encyclopedia of Genes and Genomes pathway analysis of differentially expressed genes shows enrichment of the proteasome pathway in Sftpc^C185G AEC2s. (H) Proteasome activity assessed as chymotrypsin-like activity in cell lysates of AEC2 isolated 7 d post-TMX. **p < 0.005, ***p < 0.0005 by two-way t test.