Fig. 6: Malat1 binds to Tead3 to inhibit Nfatc1 activity.

a Immunoblotting of Yap and Tead1-4 in B16F1, MEF, BMM, MSC, L929, and RAW264.7 cells. b, c Total Tead (b) or Tead3 (c) was immunoprecipitated from RAW264.7 cells. Tead- or Tead3-bound Malat1 was quantitated by qPCR. d Biotinylated (Btn) Malat1 fragments were synthesized in vitro (upper), incubated with V5-Tead3-overexpressing HEK293T cell lysates, and pulled down with streptavidin beads. The bound proteins were immunoblotted with a V5-specific antibody (lower). e, f Immunoblotting of NFATC1, Ctsk or TRAP, and TEAD1-4 in RANKL-treated RAW264.7 (e) or U937 (f) cells. SE short exposure, LE long exposure. g HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with S-protein beads or a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG. h, i Control and MALAT1-knockout HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (h) or S-protein beads (i) and immunoblotting with antibodies against MYC and FLAG. j, k MALAT1-knockout and Malat1-restored HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (j) or S-protein beads (k) and immunoblotting with antibodies against MYC and FLAG. l Mouse Nfatc1, Tead3, and truncation mutants. m HEK293T cells were co-transfected with MYC-Tead3 and SFB-Nfatc1 (full-length or truncated), followed by pulldown with S-protein beads and immunoblotting with antibodies against MYC and FLAG. n HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3 (full-length or truncated), followed by pulldown with a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG. o, p Luciferase activity in HEK293T cells co-transfected with Tead3, constitutively active Nfatc1 (Nfatc1-CA), Renilla luciferase, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (o) or the Ctsk promoter (p). q, r Luciferase activity in wild-type, MALAT1-knockout, and Malat1-restored HEK293T cells co-transfected with Tead3, Nfatc1-CA, Renilla luciferase, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (q) or the Ctsk promoter (r). Statistical significance in b, c, and o–r was determined by a two-tailed unpaired t test. Error bars are s.e.m. n = 3 biological replicates. The experiments in a, d–k, m, and n were repeated independently three times, yielding similar results. Source data are provided as a Source Data file.