Figure 1.
Generation of SARA FYVE domain deficient mice.
(A) Schematic diagram of the SARA genomic locus, targeting construct, and genomic locus of the resulting SARA-dFYVE mutant mice after Cre-mediated deletion. The numbered black boxes are SARA exons. The open and black arrowheads indicate loxP and FRT sites, respectively. Neor is the neomycin resistance cassette. The flanking probe used in Southern blotting and the expected fragment sizes after BamHI digestion of wild type (16.6 kb) and mutant (6.8 kb) genomic DNA are indicated. The locations of the PCR primers used to screen genotypes are shown (arrows). (B) ES cell clones with correctly disrupted alleles were confirmed by Southern blot analysis with the 5′ external probe indicated in panel A. Two independent SARA-CKO ES clones (#19 and #60) were identified. (C) Genotyping of SARA-dFYVE mutant mice by PCR using the primers (WT1 and WT2) or (KO1 and KO2) are shown in panel A. (D) Total protein lysates (0.5 mg) from MEFs were immunoprecipitated (IP) with anti-mouse SARA-N antibody and then blotted (IB) with anti-human SARA antibody. The expression of SARA1 and SARA2 proteins was completely abolished in SARAΔ/Δ mice. Western blotting with the GAPDH antibody served as the input control.
Figure 2.
SARA expression patterns in embryonic and adult mice.
(A) SARA transcripts were detected in the mouse embryos at embryonic day (E) 7.5 to E10.5 by whole-mount in situ hybridization. (B) Western blotting was performed to evaluate the expression of SARA in each adult mouse tissue. Total protein lysates (1 mg) were immunoprecipitated (IP) with anti-mouse SARA-N antibody and then blotted with anti-human SARA antibody. Adult mouse tissues are as follows: brain (lane 1), heart (lane 2), lung (lane 3), liver (lane 4), kidney (lane 5), spleen (lane 6), and skin (lane 7). Two isoform of SARA proteins (SARA1 and SARA2) were detected in each tissue except the kidney. Expression of GAPDH was used as the input control. (C) RT-PCR analysis of adult brain (lane 1) and kidney (lane 2) mRNAs was performed using the primer pair (SARA-E1-f and SARA-E2-r) for SARA1/2 transcripts. SARA1 and SARA2 transcripts were detected in adult mouse kidneys. GAPDH served as the input control.
Figure 3.
Expression of truncated SARA proteins in SARA mutant mice.
(A) Schematic diagram of the mouse SARA mRNA transcripts in WT and SARA-dFYVE mutant mice. The black boxes are SARA exons and the open arrowheads indicate loxP sites. The locations of the RT-PCR primers used to detect SARA mRNA transcripts are shown (arrows). (B) RT-PCR analysis of mouse brain total RNA, performed using the primer pair (SARA-E2-f and SARA-E17-r) for SARA1 and SARA2 transcripts and the primer pair (SARA-E1-f and SARA-E5-r) for SARA3 and SARA4 transcripts, is shown in panel A. (C) To confirm that truncated SARA proteins (SARA3 and SARA4) were expressed in SARA-dFYVE mutant mice, Total protein lysates (1 mg) from adult skin was immunoprecipitated (IP) and blotted (IB) with anti-mouse SARA-C antibody.
Figure 4.
TGF-β/Smad2 signaling is downregulated in SARA mutant mice.
(A) Serum-starved MEFs were treated with or without 4 ng/mL TGF-β1 for 30 min. The cellular locations of Smad2 and Smad3 were detected by immunofluorescence staining using Smad2 and Smad3 antibodies. TGF-β-induced nuclear translocation of Smad2 but not Smad3 was decreased in SARA mutant MEFs. (B) After cells were treated with or without 4 ng/mL TGF-β1 for 1 hour, MEF lysates were collected and analyzed by Western blot. Total Smad protein and phosphorylated Smad protein were detected by specific antibodies as indicated. (C) Quantification of Western blot results in panel B showed that mutation of SARA protein did not alter the ability of Smad2 protein phosphorylation. Although Smad2 protein levels were reduced, Smad3 expression was not altered. (D) Analysis of Smad2 expression in mouse skin by immunohistochemistry with Smad2 antibody. The controls were incubated with only the secondary antibody, as shown in the insert sections. Smad2 protein was decreased in SARAΔ/Δ skin compared with WT.
Figure 5.
Loss of FYVE domain of SARA does not affect the internalization of TGF-β receptors into the early endosome.
WT and SARAΔ/Δ MEFs were incubated at 4°C for 1 hour and then treated with 4 ng/mL TGF-β1 for 30 minutes at 37°C. Cells were fixed and stained with antibodies to endogenous EEA1, TGF-β RI (A), and RII (B). The overlap between the two signals is displayed in yellow (indicated by arrows).
Figure 6.
SARA does not participate in TGF-β-mediated cellular responses.
(A) Growth curves of MEFs were determined using the MTT assay. Cells were treated with or without 4 ng/mL TGF-β1 and stained with MTT at the times indicated. The data points are the average of three independent measurements, and the standard deviation from the mean is shown. (B) The morphology of apoptotic cells was observed by Hoechst 33342 staining. MEFs were serum-starved for 24 hours and then treated with or without 10 ng/mL TGF-β1 for 24 hours. Apoptotic cells showed condensed chromatin and a fragmented apoptotic nucleus (indicated by arrows). The percentage of apoptotic cells was counted in ten random fields for each triplicate sample. (C) MEFs were treated with or without 4 ng/mL TGF-β1 for 6 days, fixed, and stained with β-gal. The percentage of senescent cells was counted in ten random fields for each triplicate sample. (D) MEFs were serum-starved for 24 hours and then treated with or without 10 ng/mL TGF-β1 for 3 days. Cells were subjected to immunocytochemistry and Western blot analysis using an anti-α-SMA antibody. The percentage of α-SMA positive cells was counted in ten random fields for each triplicate sample. Coomassie blue stain penicillin-streptomycinserved as the loading control.
Figure 7.
SARA prevents Smurf2-induced Smad2 degradation.
(A) Smad2 RNA expression levels in WT and SARA mutant MEFs were quantified by Q-PCR. Data represent means of three independent experiments performed in triplicate. (B) Expression of Smurf2 protein in WT and SARA mutant MEFs were detected by Western blot analysis (upper panel). MEF lysates (500 µg) were immunoprecipitated (IP) with Smurf2 antibody and blotted (IB) with the indicated antibodies (lower panel). (C) WT and SARA mutant MEFs were treated with DMSO and MG132 (20 µM) for 8 hours prior to lysis. Expression levels of Smad2 protein were quantified by Western blot analysis. (D) MEF lysates (500 µg) were immunoprecipitated (IP) with Smurf2 or mSARA-N antibody and blotted (IB) with the indicated antibodies.
Figure 8.
Loss of SARA promotes skin tumor formation and malignant progression.
(A) Average number of tumors in WT and SARA mutant mice at different time points. Arrow indicates TPA withdrawal. Twenty weeks after the beginning of promotion, a significant difference in the number of papillomas per mouse between the WT and SARA mutant mice were evident (P<0.05). (B) Incidence of malignant progression in skin tumors generated 40 weeks after the beginning of promotion. SARAΔ/Δ mice showed a highly significant increase in the percentage of SCCs compared with SARA+/Δ and WT controls. Moderately- and poorly-differentiated SCCs were not found in WT mice. (C) Histological analysis of skin tumors. Papilloma (note a proliferation of hyperkeratotic stratified squamous epithelium); well-differentiated SCCs (note tumor cells destroy the basement membrane and invade the dermis); moderately-differentiated SCCs (note cells are markedly irregular in shape and size, distinct nuclear pleomorphism and mitotic activity); and poorly-differentiated SCCs (note immature cells predominate, with numerous atypical mitosis, and minimal keratinization) are shown.