Journal: Cell Death & Disease

Article Title: USP24-dependent STAT2 stabilization mediates physiologic and pathologic bone formation

doi: 10.1038/s41419-026-08888-7

Figure Lengend Snippet: 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 10 11 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).

Article Snippet: After sections were incubated with antibodies specific to USP24 (1:100, Novus Biologicals, NBP1-82942) or STAT2 (1:100, Abclonal, A14995) for 40 min at 37 °C, and secondary antibodies for 20 min at 37 °C.

Techniques: Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Binding Assay, Generated, Ubiquitin Proteomics, Expressing, Injection, Cell Culture, Activity Assay, Gene Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: USP24-dependent STAT2 stabilization mediates physiologic and pathologic bone formation

doi: 10.1038/s41419-026-08888-7

Figure Lengend Snippet: 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-ER T2 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-ER T2 mice were treated with i.p. injection of tamoxifen for 5 days and 5 days later, 5 × 10 12 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 × 10 12 vg/kg of rAAV9.egfp carrying amiR-Ctrl or amiR-Usp24 was injected into 8-week-old Acvr1 (R206H)Fl ;Cre-ER T2 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.

Article Snippet: After sections were incubated with antibodies specific to USP24 (1:100, Novus Biologicals, NBP1-82942) or STAT2 (1:100, Abclonal, A14995) for 40 min at 37 °C, and secondary antibodies for 20 min at 37 °C.

Techniques: Staining, Injection, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Micro-CT, Negative Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: USP24-dependent STAT2 stabilization mediates physiologic and pathologic bone formation

doi: 10.1038/s41419-026-08888-7

Figure Lengend Snippet: 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-ER T2 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 ( Acvr1 WT ) or Acvr1 (R206H)Fl ;Prrx1-Cre femurs, transduced with 5 × 10 10 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 ( Acvr1 WT ) or Acvr1 (R206H)Fl ;Prrx1-Cre femurs were transduced with 5 × 10 10 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 × 10 12 vg/kg of rAAV9.egfp carrying amiR-Ctrl or amiR-Stat2 was injected into 8-week-old Acvr1 (R206H)Fl ;Cre-ER T2 mice as described in Fig. , 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).

Article Snippet: After sections were incubated with antibodies specific to USP24 (1:100, Novus Biologicals, NBP1-82942) or STAT2 (1:100, Abclonal, A14995) for 40 min at 37 °C, and secondary antibodies for 20 min at 37 °C.

Techniques: Staining, Injection, Quantitative RT-PCR, Isolation, Transduction, Cell Culture, Activity Assay, Western Blot, Micro-CT, Two Tailed Test