(2007) Development 134, 2511C2519 [PMC free of charge article] [PubMed] [Google Scholar] 10. as a/the target for the inhibitory effects of Pin1 on muscle mass differentiation. We display that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle mass cells, both and and and that this interaction requires a 77-amino acid region of MEF2C immediately adjacent to the DNA binding and dimerization website. Relating to tandem mass spectrometry analysis of the MEF2C protein purified from muscle mass cells, two phosphoserine residues, Ser98 and Ser110, are present within this region. Mutation of these residues abolishes Pin1/MEF2C connection. Importantly, Pin1 overexpression negatively modulates MEF2C protein stability and activity as well as the ability of MEF2C to cooperate with MyoD to activate myogenic conversion of 10T1/2 fibroblasts. Taken together, these findings imply that Pin1 is definitely a novel bad regulator Itga4 of skeletal muscle mass terminal differentiation, a function that can be explained partly from the inhibition of stability and activity of MEF2C. EXPERIMENTAL Methods Plasmids pGL3(desMEF2)3, pRSV-gal pFLAG-MEF2C, pcDNAI/Amp/MEF2C, and pGEX-Pin1 have been explained previously (11, 12). The pcDNA-HA-Pin1 manifestation vector was generated by subcloning a PCR product of Pin1 cDNA into the pcDNA-HA-HDAC4 vector (13) after removal of the cDNA encoding HDAC4 (BamHI/EcoRI restriction). The pFLAG-MEF2C manifestation vectors bearing deletions and point mutations on Ser98, Ser110, Ser240, and Ser388, the pcDNAI/Amp/MEF2C 4SA, the pCDNA-HA-Pin1-C113A, and the pGEX-Pin1-W34A mutant plasmids were acquired by mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene). pFLAG-MEF2C-YN and pHA-Pin1-YC were acquired by cloning the PCR products of Pin1 and MEF2C cDNAs, respectively, in the pBiFC-YN and pBiFC-YC vectors. Viral vectors pLKO-puro encoding shRNAs against mouse Pin1 or a control sequence were purchased from Sigma-Aldrich. Viral vectors encoding HA-Pin1 and HA-Pin1 C113A were generated L-685458 by cloning the respective cDNAs in the pRRL-PGK-GFP transfer vector. The primers utilized for the PCR and mutagenesis reactions are available in the supplemental material. Cell Tradition and Transfection The C2C7 murine muscle mass cells, a L-685458 L-685458 subclone of the C2 muscle mass cell collection (14), have been previously explained (15). C2C7 cells were cultivated in advanced Dulbecco’s altered Eagle’s medium (A-DMEM; Invitrogen) comprising 10% fetal bovine serum (FBS; Invitrogen) (growth medium) at low denseness and, when approaching confluence, induced to differentiate with DMEM (Euroclone), 2% horse serum (Hyclone) (differentiation medium). COS1 simian kidney cells, C3H 10T1/2 mouse fibroblasts, and human being embryonic kidney (HEK) 293T cells were managed in DMEM comprising 10% FBS. Cells were transfected using the lipid-based Lipofectamine Plus reagent (Invitrogen). HEK 293T cells were transfected using the standard calcium phosphate precipitation method (16). A myogenic conversion assay of C3H 10T1/2 cells was performed as reported previously (11). Immunofluorescence and Bimolecular Fluorescence Complementation (BiFC) Assay Immunostaining of C2C7 cells cultured in L-685458 40-mm Petri dishes was performed as explained previously (17). The following primary antibodies were used: mouse M2 monoclonal anti-FLAG (F3165; Sigma-Aldrich); rabbit polyclonal anti-HA (H6908; Sigma-Aldrich), and mouse monoclonal anti-myosin weighty chain (MyHC) (MF20 Developmental Studies Hybridoma Lender). Secondary antibodies used were goat anti-mouse IgG rhodamine-conjugated (Pierce), goat anti-rabbit IgG L-685458 amino methylcoumarin acetate-conjugated (Dako). Nuclei were stained with Hoechst 33342 (Sigma-Aldrich). For the BiFC assay, C2C7 cells were transfected with the indicated plasmids, and 36 h after transfection, they were incubated for 30 min at 30 C to enhance the fluorophore maturation of the yellow fluorescent protein (YFP). All samples were examinated inside a Zeiss Axioskop 40 fluorescence microscope equipped with an Axiocam HRC video camera for image acquisition. Quantitative estimations of nuclei present in MyHC-positive cells were performed using the Cell Counter plugin of Image J (available on the National Institutes of Health Internet site) by analyzing at least three fields for each sample (3 103 nuclei). This experiment.