Figure 6

The ALK6 (Activin Receptor-Like Kinase 6) mutant R371Q is a constitutively active BMP type 1 receptor that is hyper-responsive to BMP ligands. C2C12BRA cells were stably transfected encoding a luciferase downstream of the BMP response element promoter (BRE-LUC), as well as either wild-type ALK6 (WT), a constitutively active ALK6 (CA), or R371Q ALK6. The ALK6 mutant R371Q (which results from the rs34970181_A variant) had increased baseline BMP signaling activity compared to WT ALK6 (A, eight replicates per group). Compared to WT ALK6, the R371Q ALK6 variant had increased BMP signaling in response to BMP2 (B, five replicates per group). Compared to WT ALK6, expression of the R371Q ALK6 variant in HepG2 cells resulted in increased BMP-induced DGAT2 (diacylglycerol O-acyltransferase 2) mRNA levels (C, six replicates per group). Equal levels of expression of WT and R371Q ALK6 was confirmed by Western blot using antibodies detecting the HA epitope tag (D). GAPDH was used as a loading control. Immunofluorescence staining of HepG2 cells for pSMAD1/5/8 and DGAT2 transfected with wild-type ALK6 or R371Q ALK6 (E) and quantified using quantitative microscopy (F, % intensity represents signal of fluorophore vs background). The comparisons for (A) were performed using a 1-way ANOVA with Sidak’s multiple comparison testing and, for (B,C,F), two-tailed Student’s t test was used.