Fig. 3

BMPR1A^R443C does not alter ACVR1^R206H‐induced BMP signaling in ACVR1^R206H HEK 293T cells or Tg(R206Ha) zebrafish, but BMPR1A knockdown in ACVR1^R206H HEK 293T cells decreases BMP signaling. (A) pSMAD 1/5/9 levels normalized to GAPDH in nontransfected ACVR1 ^R206H HEK 293T cells stimulated with BMP4 or AA. The fold‐change between NS and BMP4‐ or AA‐stimulated cells is 3.69 and 8.81, respectively, for ACVR ^R206H HEK 293T cells. p‐values are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (B) pSMAD 1/5/9 levels normalized to GAPDH in nontransfected ACVR1 ^R206H HEK 293T cells after transient transfection with siRNA targeting BMPR1A and stimulation with either BMP4, activin A (AA), or nonstimulated. N ≥ 5 for each condition. p‐values are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (C) Representative Western blots showing pSMAD 1/5/9 levels normalized to GAPDH loading control in HEK 293T ACVR1 ^R206H cells after transient transfection with siRNA targeting BMPR1A and stimulation with either BMP4 or AA. (▲ = duplicate NT/AA sample repeated across all 3 membranes for normalization); n ≥ 5 replicates for each condition. Horizontal boxes denote Western blots that were run on the same membrane but cut between 50 kD and 37 kD prior to separate pSMAD 1/5/9 and GAPDH antibody probing. p‐values are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (D) pSMAD 1/5/9 levels normalized to GAPDH and BMPR1A‐HA protein tag in ACVR1 ^R206H HEK 293T cells after transient transfection with BMPR1A ^WT or BMPR1A ^R443C plasmids; n ≥ 5 biological replicates for each condition. (E) Representative Western blot demonstrating pSMAD 1/5/9 levels normalized to GAPDH and BMPR1A‐HA protein tag in ACVR1 ^R206H HEK 293T cells after transient transfection with BMPR1A ^WT or BMPR1A ^R443C plasmids (representative of n = 6 biological replicates). Horizontal boxes denote Western blots that were run on the same membrane but cut between 50 kD and 37 kD prior to pSMAD 1/5/9 and GAPDH antibody probing. p‐values are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (F) (Columns 1–4) Percent of dorsalized embryos in WT (non‐Tg(ACVR1‐R206Ha)) zebrafish in a bmpr1aa; bmpr1ab mutant background after injection with either BMPR1A ^WT (90 ng/μL) or BMPR1A ^R443C (90 ng/μL) human mRNA. Kruskal‐Wallis test was performed and, statistical significance observed between columns 6 + 8 (p = 0.0004 (Columns 1–4, n = 58, 63, 60, 105 embryos, respectively). (Columns 5–8) Percent of ventralized embryos in Tg(ACVR1‐R206Ha) zebrafish after injection with human BMPR1A ^WT or BMPR1A ^R443C mRNA). Kruskal‐Wallis test was performed, statistical significance observed between columns 2 and 4 (p < 0.0001) (Columns 5–8, n = 50, 71, 39, and 71 embryos, respectively). (G,H) Representative dorsalized non‐Tg(ACVR1‐R206Ha) embryos in bmpr1aa; bmpr1ab mutant background following BMPR1A ^WT (90 ng/μL) or BMPR1A ^R443C (90 ng/μL) human mRNA injection. Parental genotypes are indicated above in F. BMPR1A ^WT / ^R443C mRNAs were injected into separate clutches, and uninjected controls were scored from each clutch. Transgenic‐positive and transgenic‐negative embryos were sorted via mCherry fluorescence.