The mRNA level of was determined by RT-qPCR and normalized to 0.05 vs. induction, the detailed mechanisms that diminish Id3 function and cause the cells to withdraw from the cell cycle are unknown. Induction of myoblast differentiation resulted in decreased expression of Id3 and increased expression of XBP1u, and XBP1u accelerated proteasomal degradation of Id3 in C2C12 cells. The expression levels of the cyclin-dependent Chitinase-IN-2 FKBP4 kinase inhibitors p21, p27, and p57 were not Chitinase-IN-2 increased after differentiation induction of XBP1-knockdown C2C12 cells. Moreover, knockdown of Id3 rescued myogenic differentiation of XBP1-knockdown C2C12 cells. Taken together, these findings provide evidence that XBP1u regulates cell cycle exit after Chitinase-IN-2 myogenic differentiation induction through interactions with Id3. To the best of our knowledge, this is the first report of the involvement of XBP1u in myoblast differentiation. These results indicate that XBP1u may act as a regulator of myoblast differentiation under various physiological conditions. the Chitinase-IN-2 proteasomes of C2C12 cells. Knockdown of Id3 rescued myogenic differentiation of XBP1-knockdown C2C12 cells. These obtaining suggest that XBP1u plays an important role in switching from the undifferentiated to differentiated state by targeting Id3 for degradation. Materials and Methods Reagents Dulbeccos altered Eagle medium (DMEM) was purchased from Invitrogen (Grand Island, NY, United States). Fetal bovine serum (FBS) was purchased from EQUITECH-BIO (Cotton Gin Lane, TX, United States). Horse serum was obtained from Thermo Scientific (Waltham, MA, United States). Precast, 4C20% Mini-PROTEAN TGX gels and Polyvinylidene fluoride (PVDF) membranes were obtained from Bio-Rad (Hercules, CA, United States). MG132, Cycloheximide (CHX), Id3 siRNA, and non-targeting control siRNA were purchased from Sigma-Aldrich (Saint Louis, MO, United States). All other compounds were purchased from Nacalai Tesque (Kyoto, Japan). Antibodies For immunoblotting analysis, the following antibodies were used: anti-Myosin 4 (MF20) monoclonal antibody (Thermo Scientific), anti-Flag (catalog number: PM020) rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-XBP1 rabbit polyclonal antibody (catalog number: sc-7160), anti-Id3 (catalog number: sc-56712), anti-p21 (catalog number: sc-6246), anti-cyclin D1 (catalog number: sc-8396) mouse monoclonal antibody (Santa Cruz Biotechnology), anti–tubulin (catalog number: PM054), anti-mouse or anti-rabbit IgG HRP-linked whole Ab (GE Healthcare, Chicago, IL, United States). Plasmids The XBP1u expression plasmid was generated using standard DNA techniques. Mouse cDNA encoding Id3 (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_008321″,”term_id”:”162417969″,”term_text”:”NM_008321″NM_008321) was amplified from total RNA of skeletal muscle tissue by RT-PCR using a sense primer (5-TCCCTCTCTATCTCTACTCTCCAAC-3) and an antisense primer (5-AGTCCCAGGGTCCCAAGC-3). Flag-Id3 was produced by cloning into a pFLAG-CMV expression vector (Sigma-Aldrich). The nucleotide sequences of PCR products and construct were verified by sequencing. The sequence was analyzed using the basic local alignment search tool (BLAST). The pcDNA3.1(-) plasmid encoding XBP1u was a kind gift from Dr. Ann-Hwee Lee, Ph.D. (Regeneron Pharmaceuticals, Inc., Tarrytown, NY, United States). Cell Culture C2C12 mouse myoblast cell line (DS Pharma Biomedical, Osaka, Japan) and previously generated XBP1-knockdown cell lines were cultured as described previously (Tokutake et al., 2019). Briefly, the cells were cultured in Dulbeccos altered Eagles medium (DMEM) supplemented with 10% fetal bovine serum under an atmosphere of 5% CO2/95% air at 37C. Primary mouse myoblasts were isolated from 4 week-old C57bl/6J mice (Japan SLC, Hamamatsu, Japan). Undifferentiated myoblasts were maintained in growth medium consisting of DMEM supplemented with 20% fetal bovine Chitinase-IN-2 serum and 2 ng/mL basic fibroblast growth factor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). To induce differentiation into myotubes, cells were shifted to DMEM supplemented with 2% horse serum. To assess degradation of Id3 by XBP1u, C2C12 cells were co-transfected with p cDNA3.1(-)-XBP1u and a Flag-Id3 construct using Lipofectamine 2000 reagent (Invitrogen Corporation, Carlsbad, CA, United States) in accordance with the manufacturers protocol. At 24 h post transfection, the proteasome inhibitor.