Fig 1: USP24 controls STAT2 protein stability in osteoblasts.A C3H10T1/2 cells were transfected with either vector control or Flag-USP24, and 48 h later, mRNA levels (left, n = 4) and protein levels (right) of USP24 were determined by qRT-PCR and immunoblotting, respectively. B Enrichment scores of putative USP24-binding proteins generated by WebGestalt pathway analysis. C Flag-USP24 and His-ubiquitin were transfected into HEK293T cells along with STAT1, STAT2, or STAT3. After 48 h of transfection, cells were treated with 10 μM MG132 for 6 h, lysed, pulled down with Ni-NTA agarose, and immunoblotted with the indicated antibodies. D Flag-USP24 and HA-ubiquitin were transfected into C3H10T1/2 cells. 48 h later, cells were treated with 10 μM MG132 for 6 h, lysed, pulled down with Flag-conjugated agarose, and immunoblotted for STAT2. STAT2 protein levels (E) and mRNA levels (F, n = 4) in shCtrl- or shUsp24-expressing mouse COBs. G 1011 GCs of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24 were injected into P0 neonates, and 28 days later, STAT2 protein levels in the tibia were assessed by immunoblotting. H shCtrl or shUsp24-expressing mouse COBs were treated with 10 μM MG132 for 6 h and immunoblotted for STAT2. shCtrl or shStat2-expressing mouse COBs were cultured under osteogenic conditions. Six days later, ALP activity (I, n = 9) and osteoblast gene expression (J, n = 4) were examined. Data are representative of three (A, C–J). A two-tailed unpaired Student’s t test for comparing two groups (A, F, I, J; error bars, SD of biological replicates).
Fig 2: USP24 induces heterotopic ossification in vivo FOP mouse model.A H&E-stained sections (left) and IHC (middle and right) for USP24 in human tissue with heterotopic ossification (HO). Scale bar, 50 μm (left and middle) or 20 μm (right). B 8-week-old Acvr1(R206H)Fl; Cre-ERT2 mice were treated with intraperitoneal (i.p.) injection of tamoxifen for 5 days, and 3 days later, the pinch injury/cardiotoxin (CTX) injection was introduced to the gastrocnemius muscle. 24 h after the injury, the tibial muscle was dissected, and Usp24 mRNA levels were assessed by qRT-PCR (n = 4). C Schematic diagram showing experimental strategy and plan. D 8-week-old Acvr1(R206H)Fl;Cre-ERT2 mice were treated with i.p. injection of tamoxifen for 5 days and 5 days later, 5 × 1012 vg/kg of rAAV9.egfp was injected into the gastrocnemius muscle (i.m.), followed by CTX injection and pinch injury. 28 days later, EGFP expression was assessed by fluorescence microscopy. Scale bar, 100 μm. HO, heterotopic ossification; BM, bone marrow; M, muscle. E 5 × 1012 vg/kg of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24 was injected into 8-week-old Acvr1(R206H)Fl;Cre-ERT2 mice as described in Fig. 4C, and Usp24 mRNA levels were assessed by qRT-PCR (n = 6 for male and n = 5 for female). F Incidence (%) of HO-positive mice. The number of HO-positive mice is shown as a proportion of the total mice in each group (n = 12 per group). In males, the incidence was 10/12 (83.3%) for the amiR-Ctrl- and 6/12 (50.0%) for the amiR-Usp24-treated group (p = 0.193). In females, 11/12 (91.7%) for the amiR-Ctrl- and 6/12 (50.0%) for the amiR-Usp24-treated group (p = 0.068). HO was detected by radiography and quantified by micro-CT. Representative radiographic images (G), quantified HO bone volume (BV, H, n = 5), and 3D reconstruction images (I) were shown. Scale bar, 1 mm (I). HO was assessed by H&E staining (J) and IHC for STAT2 (K) in rAAV-treated femurs. Scale bar, 250 μm (J, upper), 100 μm (J, lower), or 20 μm (K). IgG was used as a negative control. Data are representative of three (A, D, G, I–K). A two-tailed unpaired Student’s t test for comparing two groups (B, E, H; error bars, SD of biological replicates). For Fig. 4F, statistical significance was determined by Fisher’s exact test.
Fig 3: A USP24/STAT2 signal axis is required for HO formation.A H&E-staining (left) and IHC for STAT2 (right) in human HO tissue. Blue arrows indicate bone-lining cells; Orange arrows indicate bone-embedded osteocytes. Scale bar, 20 μm. B 8-week-old Acvr1(R206H)Fl; Cre-ERT2 mice were treated with i.p. injection of tamoxifen for 5 days, and 5 days later, the pinch injury and CTX injection were introduced to the gastrocnemius muscle. 24 h after injury, the tibial muscle was dissected, and Stat2 and Ifi204 mRNA levels were assessed by qRT-PCR (n = 4). C Schematic diagram illustrating the mechanisms by which USP24 promotes osteoblast differentiation through STAT2 stabilization. Prrx1+ osteogenic progenitors isolated from 4-week-old Prrx1-Cre (Acvr1WT) or Acvr1(R206H)Fl;Prrx1-Cre femurs, transduced with 5 × 1010 GCs of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24, and cultured in osteogenic induction medium (OIM) with or without activin A (50 ng/ml). ALP activity (D, n = 6) and mRNA levels of osteoblast genes (E, n = 4) were examined at day 6. Protein levels of USP24, phospho-SMAD1/5, and STAT2 were determined by immunoblotting at day 1 of osteogenic differentiation (F). Prrx1+ osteogenic progenitors isolated from 4-week-old Prrx1-Cre (Acvr1WT) or Acvr1(R206H)Fl;Prrx1-Cre femurs were transduced with 5 × 1010 GCs of rAAV9.egfp carrying amiR-Ctrl or amiR-Stat2 and cultured in OIM with or without activin A (50 ng/ml). ALP activity (G, n = 6) and mRNA levels of osteoblast genes (H, n = 4) were examined at day 6. I 5 × 1012 vg/kg of rAAV9.egfp carrying amiR-Ctrl or amiR-Stat2 was injected into 8-week-old Acvr1(R206H)Fl;Cre-ERT2 mice as described in Fig. 4C, and Stat2 mRNA levels were assessed by qRT-PCR (n = 4). HO formation was assessed by micro-CT. 3D reconstruction images (J) and quantified HO bone volume (BV, K n = 8 for amiR-Ctrl and n = 6 for amiR-Stat2) were shown. Scale bar, 1 mm (J). Data are representative of three (A, F, J). A two-tailed unpaired Student’s t test for comparing two groups (B, D, E, G–I, K; error bars, SD of biological replicates).
Supplier Page from Sino Biological, Inc. for Human STAT2 Gene ORF cDNA clone expression plasmid, N-Myc tag