Fig 1: BYL719 does not reduce ACVR1 kinase activity.(A) A schematic depiction of nano-bioluminescence resonance energy transfer (nanoBRET) target engagement assays. TGF-β receptors–Nanoluciferase fusion proteins are expressed in COS-1 cells and an ATP-like tracer analog in close proximity with the Nanoluciferase donor allows energy transfer from the Nanoluciferase donor to the fluorescent tracer acceptor. An ATP analog molecule (type I inhibitor) will compete with the fluorescent tracer, impairing close proximity donor–acceptor and reducing the nanoBRET ratio. (B) The nanoBRET emission spectra consist of the Nanoluciferase donor (460 nm) and the fluorescent acceptor (610 nm). The nanoBRET ratio is shown as milliBRET units (mBU) by dividing the acceptor emission by the donor emission times 1000. (C) NanoBRET target engagement analyses of ACVRL1, ACVR1, ACVR1R206H, BMPR1A, ACVR1B, TGFBR1, TGFBR2, ACVR2A, ACVR2B, and BMPR2 testing 1 or 10 µM BYL719 with n = 4. As controls, LDN193189 (0,5 µM), SB431542 (10 µM), and ML347 (10 µM) were used. Data are shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test. (D) Casein phosphorylation by ACVR1R206H kinase. Phosphorylation was performed in the presence of ACVR1R206H kinase and increasing concentrations of the PI3Kα inhibitor BYL719. Quantification of kinase activity. Data are shown as mean ± SD (n = 4 independent experiments). One-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 4—source data 1.Original files for autoradiographies displayed in Figure 4D. Figure 4—source data 2.PDF file containing original autoradiographies for Figure 4D, indicating the relevant bands and treatments.
Fig 2: Analysis of Acvr1 gene expression by qPCR in bone marrow-derived mesenchymal stem cells (BM-MSCs) from Pik3cafl/fl mice infected with virus expressing wild-type Acvr1 (WT) or Acvr1R206H (RH).(A) The endogenous expression level of the Acvr1 gene in mock-transfected BM-MSCs is shown as a dotted horizontal line. Data are shown as mean ± SD (n = 12 per group). Unpaired t-test between transfected groups. (B) Gene expression analysis of Pik3caα in BM-MSCs from Pik3cafl/fl mice, infected with virus expressing wild-type Acvr1 (WT) or Acvr1R206H (RH) and/or Cre co-infection. Data are shown as mean ± SD (n = 3 per group). ***p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. (C) mRNA expression of canonical (Id1 and Sp7), non-canonical (Ptgs2) target genes, and activin A (Inhba) in BM-MSCs Pik3cafl/fl transfected with Acvr1 (wild type or R206H) with or without Cre recombinase. Cells were NT (not treated) or treated with BYL719 (2 µM) and/or activin A (2 nM). Expression data were normalized to those of control cells which were transfected only with Acvr1 WT without any treatment, shown as a dotted horizontal line. Asterisks (*) refer to the differences between different conditions of Acvr1 RH cells compared to control cells. Hash signs (#) refer to the differences between different conditions of Acvr1 RH cells compared to Acvr1 RH cells without Cre recombinase and treated with activin A. Data are shown as mean ± SD (n = 6 per group). * or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.
Fig 3: BYL 719 inhibits ossification and osteoblast differentiation processes in human ACVR1-R206H MSCs.(A) Schematic depiction of the experimental setup, involving bulk RNA sequencing of human MSC-ACVR1WT and ACVR1R206H upon overnight starvation, 30-min pre-treatment with or without 1 µM BYL719 and with or without 1 hr activin A (50 ng/ml) or BMP6 (50 ng/ml) stimulation. (B) The top 10 most significant gene ontology (GO) terms using all (up- and downregulated) differentially expressed genes between cells expressing ACVR1-WT and ACVR1-R206H under control conditions. Ossification (GO:0001503) and osteoblast differentiation (GO:0001649) were detected within the top 10 of the differentially regulated biological processes. (C) Table of GO terms ossification and osteoblast differentiation upon GO enrichment analysis in all tested conditions ACVR1WT or ACVR1R206H cells, stimulated with BMP6 or activin A and treated with BYL719. Statistical significance upon GO enrichment analysis, count of total differentially expressed genes (DEGs), and their classification as up- or downregulated DEGs are detailed for each comparison. (D) Gene set enrichment analysis (GSEA) with the groups RH_ActA_BYL vs RH_ActA, showing the enrichment plots of the gene ontology sets ossification (GO: 0001503) and osteoblast differentiation (GO:0001649). (E) A heatmap of the top 40 most relevant genes within the ossification GO geneset derived from the leading-edge subset of the enrichment plot (n = 4 per group). Sample names are detailed as receptor (R206H) ligand (AA, Act A, Activin A)_inhibitor (BYL719, if present)_replicate#.
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