Fig 1: JAB0505 profoundly exacerbates HO and extends the period of lesional growth in FOP mice.(A) Representative µCT images of HO in Acvr1FLEx(R206H)/+; CAG-CreERT2 mice 20 days after cardiotoxin-induced injury of the gastrocnemius muscle (Untreated, n = 3; JAB0505, n = 4). (B) Representative µCT images of HO (pseudocolored green) in Acvr1tnR206H/+; Tie2-Cre mice 21 days after pinch injury of the gastrocnemius muscle (Untreated, n = 11; JAB0505, n = 10). (C) Quantification of HO volumes as a function of time after muscle pinch injury of Acvr1tnR206H/+; Tie2-Cre mice. Untreated, n = 11; JAB0505 (10 mg/kg), n = 6. Error bars represent ±SEM. ****P = 0.0001 by 2-way ANOVA with Sidak’s multiple-comparison test. (D) Paired single transverse slice and 3D reconstructed µCT images of the distal hind limb of Acvr1tnR206H/+; Tie2-Cre mice at the indicated times after hind limb muscle pinch injury with and without administration of JAB0505. Mineralized bone in the slice images is pseudocolored green. Radio-opaque lesional tissue below the threshold set for quantification of mineralized bone (white arrows in day 14 slices) is extensive at day 14 in JAB0505-treated mice. Mineralized bone in day 14 slices is barely visible at this magnification. HO volumes are given for images prior to day 35. The tibia and fibula are labeled with asterisks in the day 14 slices. Pelvic bones present in day 21, 28, and 35 slices of JAB0505-treated mice are denoted with arrowheads. To avoid confusion with HO, the baculum present in some images was removed by segmentation.
Fig 2: JAB0505 functions as an agonist of ACVR1(R206H).(A) Osteogenic differentiation of monolayer R206H-FAP cultures, as assessed by ALP staining (purple), and (B) chondrogenic differentiation of micromass cultures assessed by Alcian blue staining. ActA-mAb was used at 1 µg/mL (7 nM) and JAB0505 was used at 10 µg/mL (~70 nM). (C) Western blot of phosphorylated SMAD1/5/8 (p-SMAD1/5/8) in wild-type (WT) and R206H-FAPs (R206H). ß-Actin was used as a loading control. (D) µCT of the distal hind limb of Acvr1tnR206H/+; Tie2-Cre mice on day 21 after injury. At the time of muscle injury, mice were treated with ActA-mAb (10 mg/kg) alone or ActA-mAb with JAB0505 (10 mg/kg). HO is pseudocolored green, and quantification is shown. ActA-mAb, n = 6; JAB0505 plus ActA-mAb, n = 6. Error bars represent ±SD. ****P < 0.0001 by 2-tailed, unpaired t test. (E) µCT images of the distal hind limb 21 days after transplantation of R206H-FAPs into the injured gastrocnemius of SCID hosts. ActA-mAb (10 mg/kg) and JAB0505 (10 mg/kg) were administered at the time of transplantation. HO is pseudocolored green and quantified, with error bars representing ±SD. Untreated, n = 10; JAB0505, n = 16; ActA-mAb, n = 6; JAB0505 plus ActA-mAb, n = 8.
Fig 3: JAB0505 treatment causes a sustained increase in R206H-FAPs following skeletal muscle injury of FOP mice.(A) 3D tomographic bioluminescence source reconstruction following muscle pinch injury of Acvr1tnR206H/+; R26Luc/+; Tie2-Cre FOP mice, with and without administration of JAB0505. Paired images show µCT alone (left panel) and µCT combined with the corresponding 3D bioluminescence reconstruction (right panel). The same mouse is shown from days 3 to 21. Bioluminescence reconstruction was not performed on day 21 due to the dampening effect of dense bone on luminescent output. (B) Graphical representation of bioluminescent population dynamics of Tie2+ cells from Acvr1tnR206H/+; R26Luc/+; Tie2-Cre mice following pinch injury. Untreated, n = 16; JAB0505, n = 10. Error bars represent ±SEM. ***P = 0.001, ****P = 0.0001 by 2-way ANOVA with Sidak’s multiple-comparison test. (C) Flow cytometry analysis to determine R206H-FAP cell number in injured distal hind limb muscles of Acvr1tnR206H/+; R26NG/+; Tie2-Cre mice that were either untreated (day 5, n = 4; day 10, n = 9) or administered JAB0505 at 10 mg/kg (day 5, n = 4; day 10, n = 10). Error bars represent ±SD. **P = 0.01 by 2-tailed, unpaired t test with Welch’s correction.
Fig 4: JAB0505 lowers the injury threshold necessary to cause HO in Acvr1tnR206H/+; Tie2-Cre FOP mice.µCT images of the distal hind limbs of 4 Acvr1tnR206H/+; Tie2-Cre FOP mice (numbered 1–4) at the indicated time points after injection of 50 µL of 2.5% methylcellulose into the tibialis anterior muscle, with and without administration of 10 mg/kg JAB0505 (n = 2 mice, 4 injected limbs, for each group). HO is pseudocolored green. A lateral view of the right hind limb of each mouse is shown. Contralateral hind limbs (not shown) received equivalent injuries and the extent of HO was comparable.
Fig 5: Differential expression of ALK2 and ALK3 following LR-AMH treatment is associated with the expression of cell survival and apoptosis markers in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN ovarian cancer cells. Quantification of the western blot analysis of AMH signaling (pSMAD1/5), ALK2 and ALK3 expression, apoptosis induction (cleaved caspase-3 and PARP) and pAKT levels following incubation with 1.6 and 25 nM LR-AMH for 6 h. Data are presented as the fold-change relative to control (no LR-AMH) by bars corresponding from left to right to 0, 1.6 and 25 nM for each protein expression. n>3. Raw data are presented in Fig. S5. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; p, phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase.
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