Journal: The EMBO Journal

Article Title: GNAS/PKA signaling promotes aberrant osteochondral differentiation of Gli1 + tendon sheath progenitors

doi: 10.1038/s44318-025-00553-7

Figure Lengend Snippet: ( A , B ) Representative immunofluorescence images ( A ) and statistical analysis ( B ) of SOX9 + region in injured site from tenotomized mice following vehicle and NF449 treatment ( n = 5 per group). *** P = 8.94e-4. Scale bar, 10 μm. ( C , D ) Representative immunofluorescence images ( C ) and statistical analysis ( D ) of RUNX2 + region in injured site from tenotomized mice following vehicle and NF449 treatment ( n = 5 per group). * P = 1.37e-2. Scale bar, 10 μm. ( E , F ) Representative Safranine O staining images ( E ) and statistical analysis ( F ) of Safranine O + region in injured site from tenotomized mice following vehicle and NF449 treatment ( n = 5 per group). ** P = 1.85e-3. Scale bar, 100 μm. ( G , H ) Representative microCT images ( G ) and statistical analysis ( H ) of HO volume in injured site from tenotomized mice following vehicle and NF449 treatment ( n = 5 per group). *** P = 1.39e-4. ( I , J ) Representative Safranine O staining images ( I ) and statistical analysis ( J ) of Safranine O + region in injured site from tenotomized Gli1-CreER T2 and Gli1-CreER T2 ; Prkaca f/f mice ( n = 5 per group). *** P = 1.18e-4. Scale bar, 100 μm. ( K , L ) Representative microCT images ( K ) and statistical analysis ( L ) of HO volume in injured site from tenotomized Gli1-CreER T2 and Gli1-CreER T2 ; Prkaca f/f mice ( n = 5 per group). *** P = 5.11e-4. ( M , N ) Representative Safranine O staining images ( M ) and statistical analysis ( N ) of Safranine O + region in injured site from tenotomized Gli1-CreER T2 ; Acvr1 R206H/+ mice following vehicle and NF449 treatment ( n = 5 per group). **** P = 1.5e-7. Scale bar, 100 μm. ( O , P ) Representative microCT images ( O ) and statistical analysis ( P ) of HO volume in injured site from tenotomized Gli1-CreER T2 ; Acvr1 R206H/+ mice following vehicle and NF449 treatment ( n = 5 per group). **** P = 3.87e-6. ( Q , R ) Representative Safranine O staining images ( Q ) and statistical analysis ( R ) of Safranine O + region in injured site from tenotomized Gli1-CreER T2 ; Acvr1 R206H/+ and Gli1-CreER T2 ; Acvr1 R206H/+ ; Prkaca f/f mice at 7 dpi ( n = 5 per group). **** P = 6.2e-7. Scale bar, 100 μm. ( S , T ) Representative microCT images ( S ) and statistical analysis ( T ) of HO volume in injured site from tenotomized Gli1-CreER T2 ; Acvr1 R206H/+ and Gli1-CreER T2 ; Acvr1 R206H/+ ; Prkaca f/f mice at 14 dpi ( n = 5 per group). *** P = 1.79e-4. Data are presented as mean ± SD. All P values were determined by unpaired Student’s t test.

Article Snippet: WT and FOP mice (C57BL/6 background) that underwent tenotomy surgery were immediately treated with vehicle (saline), NF449 (MCE, 50 mg/kg), or 666-15 (MCE, 20 mg/kg) through i.p . injection every other day for 1, 2, or 4 weeks in the indicated experiments.

Techniques: Immunofluorescence, Staining

Journal: The EMBO Journal

Article Title: GNAS/PKA signaling promotes aberrant osteochondral differentiation of Gli1 + tendon sheath progenitors

doi: 10.1038/s44318-025-00553-7

Figure Lengend Snippet: ( A ) Representative HE staining showing the morphology of human tendon. Scale bar, 200 or 50 μm. ( B ) Representative immunofluorescence staining of GLI1 in human tendon. Scale bar, 10 μm. ( C ) Representative HE staining showing the morphology of HO from Ossification of the Posterior Longitudinal Ligament patients. Scale bar, 200 or 50 μm. ( D ) Representative immunofluorescence staining of GLI1 in HO from Ossification of the Posterior Longitudinal Ligament patients. Scale bar, 10 μm. ( E ) Statistical analysis of the GLI1 + cells in human normal tendon and HO lesion ( n = 3 per group). **** P = 7.88e-7. ( F , G ) Representative immunofluorescence staining ( F ) and statistical analysis ( G ) of GNAS in naive or chondrogenic tendon stem cells with or without NF449 treatment. ( n = 5 per group). **** P = 2.8e-14. Scale bar, 10 μm. ( H , I ) Representative immunofluorescence staining ( H ) and statistical analysis ( I ) of p-PKA substrate in naive or chondrogenic tendon stem cells with or without NF449 treatment. ( n = 5 per group). Data are represented as the mean ± SD. **** P = 9.77e-9. Scale bar, 10 μm. ( J , K ) Statistical analysis of SOX9 ( J , **** P = 8.91e-8) and RUNX2 ( K , **** P = 6.62e-6) in chondrogenic or osteogenic tendon stem cells with or without NF449 treatment. ( n = 6 per group). Data is presented as mean ± SD. All P values were determined by unpaired Student’s t test ( E ) and one-way ANOVA with Bonferroni post hoc test ( G , I , J , K ).

Article Snippet: WT and FOP mice (C57BL/6 background) that underwent tenotomy surgery were immediately treated with vehicle (saline), NF449 (MCE, 50 mg/kg), or 666-15 (MCE, 20 mg/kg) through i.p . injection every other day for 1, 2, or 4 weeks in the indicated experiments.

Techniques: Staining, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: GPR35 prevents drug-induced liver injury via the Gαs-cAMP-PKA axis in macrophages

doi: 10.1007/s00018-025-05751-4

Figure Lengend Snippet: GPR35 attenuates inflammatory response in macrophages through the Gαs-cAMP-PKA pathway. ( A ) Co-IP detection of GPR35 and Gαs in peritoneal macrophages stimulated with or without KA. ( B ) Levels of cAMP in peritoneal macrophages pretreated with FSK, KA, or DSCG. GPR35 agonist KA (4 mM), or DSCG (200 µg/ml) was administered 1 h before treatment with LPS. Adenylate cyclase activator FSK (10 µM) was used to stimulate cAMP in peritoneal macrophages 1 h before treatment with LPS. ( C ) Levels of TNF-α and IL-1β in supernatants from peritoneal macrophages after stimulation with LPS in the presence of FSK, KA, or DSCG. ( D ) Levels of TNF-α and IL-1β in supernatants from peritoneal macrophages with indicated pretreatment after LPS stimulation. ( E ) Levels of TNF-α and IL-1β in supernatants from peritoneal macrophages with indicated pretreatment after LPS stimulation. Gαs inhibitor NF449, AC inhibitor SQ22536, or PKA inhibitor H89 was added to the culture 30 min before treatment with GPR35 agonist KA or DSCG. The data are representative of three independent experiments and shown as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: To investigate the role of Gαs-cyclic AMP-protein kinase A (Gαs-cAMP-PKA) in the GPR35-mediated effect on macrophages, the Gαs inhibitor NF449 (25 μM, Santa Cruz Biotechnology, TEX, USA), AC inhibitor SQ22536 (50 μM, MedChemExpress, NJ, USA), or PKA inhibitor H89 (10 μM, MedChemExpress, NJ, USA), PKA inhibitor fragment [ – ] amide (10 μm, TargetMol, BOS, USA) was added to the culture 30 min prior to treatment with the GPR35 agonist.

Techniques: Co-Immunoprecipitation Assay