Fig 2: USP11 deubiquitinates SFTPC and extends its half-life. (A) BEAS-2B cells were treated with the indicated concentrations of a proteasomal inhibitor (MG132) or an inhibitor of ubiquitin activating enzyme (TAK-243) for 6 h prior to harvest. Immunoblotting was performed with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band (SFTPC/GAPDH) and are presented below the blots. (B) BEAS-2B cells were transfected with HA-USP11 or HA-USP11-shRNA and treated with MG132 for 6 h prior to harvest. A TUBEs assay was performed to assess the ubiquitination status of the SFTPC protein in mock control and HA-USP11 or HA-USP11-shRNA transduced cells. Cell lysates were immunoprecipitated with TUBEs antibodies, followed by immunoblotting with the indicated antibodies. (C) The ubiquitination and deubiquitination of endogenous SFTPC were analyzed by transfecting BEAS-2B cells with HA-USP11, HA-USP11CS, or sgRNA targeting USP11 followed by immunoprecipitation (IP) with an anti-SFTPC antibody and immunoblotting with an anti-ubiquitin antibody. The cells were treated with MG132 for 6 h prior to harvest. (D-F) The ubiquitination and deubiquitination of ectopically expressed Myc-SFTPC were analyzed by transfecting HEK293 cells with Flag-Ub along with (D) HA-USP11 and HA-USP11CS (E) HA-USP11 and sgRNA targeting USP11 (F) treatment of DUB-inhibitor PR-619 for 48 h in the HEK293 cell line prior to harvest, followed by IP with a Myc antibody and immunoblotting with an anti-Flag antibody. The relative protein expression of SFTPC-(Ub)n with respect to the input SFTPC for (C-F) was quantified using ImageJ software and is represented as (SFTPC-(Ub)n/SFTPC) below each blot. (G) The half-life of endogenous SFTPC in BEAS-2B cells. (H) The half-life of endogenous SFTPC in the presence of HA-USP11 or HA-USP11CS in BEAS-2B cells. CHX (250 μg/mL) was administered for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies. Data are presented as the mean and standard deviation of three independent experiments (n = 3). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P-values.

Fig 3: The effect of USP11 depletion on SFTPCI73T-mediated epithelial cell abnormalities and fibrosis in alveolar organoids. (A) Schematic representation of sgRNA targeting the SFTPC gene and the donor DNA design. The red arrowhead indicates the position of sgRNA1, designed to target the SFTPC gene. The blue font represents the sgRNA target sequence. A silent mutation represented in green font was introduced into the donor DNA to facilitate screening of the mutated clones by RFLP. The bold red font indicates the T to C missense mutation introduced by the HDR method. (B) Sanger sequencing analysis of the SFTPCI73T (T218C) missense mutations on the SFTPC gene. Bold letters indicate the T to C missense mutation introduced by the HDR method. (C-D) Characterization of AOs by (C) morphology by bright field and H&E staining, Scale bar: 200 μm (D) IHC staining using Pro-SFTPC and EPCAM, Scale bar: 100 μm (E-F) qRT-PCR analysis of AOs on day 45 for (E) the differentiation markers SFTPA, SFTPB, SFTPC and NKX2.1 and (F) the fibrosis markers α-SMA and COL1A1. Data are presented as the mean and standard deviation of three independent experiments (n = 3). (G-J) The effect of USP11 depletion on SFTPCI73T-mediated epithelial cell abnormalities and fibrosis in alveolar organoids was characterized by (G) H&E staining, Sirius red staining and immunostaining with COL1A1 and vimentin, Scale bar: 100 μm. (H) collagen gel contraction assay, scale bar 2 mm (I) qRT-PCR analysis of AOs using fibrosis markers COL1A1, α-SMA, and Fibronectin (J) qRT-PCR analysis of AOs using differentiation markers SFTPA, SFTPB, and SFTPC. Data are presented as the mean and standard deviation of three independent experiments (n = 3). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P-values.

Fig 4: USP11 deubiquitinates and extends the half-life of the SFTPCI73T mutant protein. (A) The predicted 3D structure of wild-type SFTPC and SFTPCI73T protein was generated using the AlphaFold tool. The structure of wild-type SFTPC was superimposed on that of its mutant at the I73T position using the UCSF chimeraX tool. (B) Western blot analysis of the basal level of transduced SFTPC and SFTPCI73T protein expression. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band (SFTPC/GAPDH). (C) Immunofluorescence analysis of the basal level of transduced SFTPC and SFTPCI73T protein expression. (D) The dose-dependent increasing effect of HA-USP11 and sgRNA-USP11 on SFTPCI73T protein levels was analyzed by western blotting. (E) Interactions between endogenous USP11 and SFTPC proteins were analyzed in BEAS-2B cells. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (F) BEAS-2B-SFTPC WT and BEAS-2B-SFTPCI73T cells were subjected to the Duolink PLA assay to analyze interactions between USP11 and SFTPC or SFTPCI73T using specific antibodies. Scale bar: 10 μm. (G) The ubiquitination status between the SFTPC and SFTPCI73T proteins in the presence of HA-USP11 or HA-USP11-shRNA. Cell lysates were immunoprecipitated with TUBEs antibodies, followed by immunoblotting with the indicated antibodies. The cells were treated with MG132 for 6 h prior to harvest. (H) The ubiquitination and deubiquitination of endogenous SFTPC and SFTPCI73T were analyzed in the presence of HA-USP11, HA-USP11CS, or sgRNA targeting USP11, followed by IP with an anti-SFTPC antibody and immunoblotting with an anti-ubiquitin antibody. The cells were treated with MG132 for 6 h prior to harvest. (I) The half-life of endogenous SFTPCI73T in BEAS-2B cells in the presence of HA-USP11 or HA-USP11CS. (J) The half-life of endogenous SFTPCI73T in BEAS-2B cells in the presence of HA-USP11 or sgRNA-USP11. CHX (250 μg/mL) was administered for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies. Data are presented as the mean and standard deviation of three independent experiments (n = 3). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P-values.