Figure 3: The mutant protein TBX1^102delK showed decreased expression level, and lost transcriptional activity.

(A,B) Real-time qPCRs show the mRNA level of the mutant TBX1^{c.303_305delGAA} was similar with that of the wild-type TBX1 in both 3T3 and C2C12 cells. (C,D) Western blots show the protein level of the mutant TBX1^102delK and wild-type TBX1. The mutant showed reduced protein level compared with the wild-type in both 3T3 and C2C12 cells. The full-length blots are presented in the Supplementary Figure S1 and S2, respectively. (E,F) show that the 3T3 or C2C12 cells were co-transfected with the 4XT/2-minP reporter and either a pcDNA3.1(+) control vector (Blank), the TBX1 wild-type construct (WT), or the mutant TBX1^{c.303_305delGAA} construct (Mut1). The results were normalized for transfection efficiency to a co-transfected pGL4.74[hRluc/TK] vector. The TBX1^102delK mutant showed significantly reduced transcription activity compared with

the wild-type protein in both 3T3 (reduced 42.4% vs. TBX1^wt, p = 0.0112) and C2C12 cells (reduced 19.9% vs. TBX1^wt, p = 0.0164). (G) Shows the C2C12 cells were co-transfected with the FGF10luc reporter and either the blank, the wild-type, or the mutant constructs (Mut1 = TBX1^102delK and Mut2 = TBX1^E129K). The results were normalized for transfection efficiency to a co-transfected pGL-TK vector. The TBX1^102delK and TBX1^E129K mutants all showed significantly reduced transcription activity compared with the wild-type protein (reduced 22.9% vs. TBX1^wt, p = 0.0050 and reduced 28.7% vs. TBX1^wt, p = 0.0112, respectively). (H) shows that significantly reduced transcriptional activity of the TBX1^102delK and TBX1^E129K mutant proteins on the WNTluc1 reporter compared with the wild-type protein in C2C12 cells (reduced 35.5% vs. TBX1^wt, p = 0.0037 and reduced 25.4% vs. TBX1^wt, p = 0.0156, respectively). (I) shows that the transcription activity of the two mutant proteins also decreased significantly on the WNTluc2 reporter in C2C12 cells (reduced 34% vs. TBX1^wt, p = 0.0259 and reduced 31% vs. TBX1^wt, p = 0.0242, respectively). The results are all shown as the mean ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; blank: pcDNA3.1(+) vector; WT: wild-type TBX1; mut1: the TBX1^102delK mutant; mut2: the TBX1^E129K mutant.