The distal arthrogryposis‐linked p.R63C variant promotes the stability and nuclear accumulation of TNNT3

Figures

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  FIGURE 1. Identification of the TNNT3 variant in affected family members with DA. (A) The pedigree of the family members affected by DA. Squares and circles represent males and females, respectively. Triangles symbolize an aborted fetus. The clinically affected individuals are indicated by filled symbols. The arrows beside the circle represent the proband. (B) and (D) Hand and foot images of the proband. The hand image (C) and its X‐ray images (E and F) of the proband's father. (G) The hand photographs of the proband's grandfather. The scripts in Figure 1H–J indicate sequencing results of the proband, I:2 and II:2, whereas the script in (K) indicates sequencing results of a healthy member of the affected family. The scripts in (L) and (M) indicate the sequencing results of amniotic fluid cultures for III:3 and III:4. (N) Protein sequences of TNNT3 from human, mouse, rat, and zebrafish were aligned to indicate a conserved Arg at residue 63 in TNNT3

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  FIGURE 2. The effect of the p.R63C variant on TNNT3 protein levels. TNNT3 protein levels in skeletal muscles from affected and non‐affected members were assessed by western blotting (TNNT3 antibody, 1:500). Representative images are shown in A, and GAPDH (1:5000) was used as an internal loading control. The relative optical density of TNNT3/GAPDH is indicated in B. TNNT3 protein levels were assessed in vitro using an anti‐Myc antibody (1:500) at different time points after transfection. Representative western blotting images in C and GAPDH (1:5000) were used as internal loading controls. The relative optical density ratio of TNNT3/GAPDH is shown in D. The results are presented as the mean ± SD, and ** p < 0.01 indicates significant differences, when compared with the corresponding wild‐type values

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  FIGURE 3. The p.R63C variant extended the half‐life of TNNT3. HEK293 cells were transfected with TNNT3 or p.R63C variant constructs, and then the cells were incubated in medium containing cycloheximide (50 μg/ml) and then chased for the indicated times. The cells were harvested at different time points, and TNNT3 protein levels were determined by western blotting using anti‐Myc antibody (1:500). Each lane resolved 40 μg of protein, and representative western blot images are shown in A. (B) The relative ratios of the optical band densities of TNNT3/GAPDH were normalized as the percentage of the ratios of the starting point as 100%. The results are presented as the mean ± SD from three independent experiments, and * p < 0.05 and ** p < 0.01 indicate significant differences, when compared to the corresponding values of TNNT3‐Myc

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  FIGURE 4. The effect of MG132 on the half‐lives of TNNT3 and the p.R63C variant. Plasmids encoding TNNT3 or the p.R63C variant were individually transfected into HEK293 cells and then incubated in medium containing either 0.1% dimethyl sulfoxide or 10 μM MG132 for 3 h. Cycloheximide (50 μg/ml) was then added to the cultures, and the cells were harvested at the indicated time points. (A) Representative western blotting images. (B) The protein levels designated by the ratios of TNNT3/GAPDH (solid circle) and TNNT3‐p.R63C/GAPDH (solid square) were normalized to the ratios of the starting point without drug treatment. The results are presented as the mean ± SD from three independent experiments, and * p < 0.05 indicates significant differences, when compared to the corresponding values of TNNT3‐Myc

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  FIGURE 5. The p.R63C variant promoted its nuclear translocation and formed aggregated puncta. (A) The plasmids encoding fusion proteins, TNNT3‐EGFP, TNNT3‐p.R63C‐EGFP, and empty vector, were transfected into HEK293 cells. After 2 days, the cultures were observed using an inverted fluorescent microscope with a GFP filter. The DAPI indicated nuclei with blue fluorescence. Scale bar, 10 µm. (B) HEK293 cells were transfected with the TNNT3‐ or p.R63C variant‐expressing vectors, and cytosolic and nuclear proteins were separated. TNNT3 protein levels in different fractions were determined with western blotting using anti‐Myc antibody (1:500). Anti‐actin (1:5000) was used as the internal control. Anti‐Histone 3 (1:5000) was used as the loading control for nuclear TNNT3. (C, D) The relative optical densities of TNNT3/actin are indicated. The results are presented as the mean ± SD from three independent experiments, and ** p < 0.01 and *** p < 0.001 indicate significant differences, when compared with the corresponding values of TNNT3‐Myc