Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: CGRP upregulates osteogenic gene and protein expression via the cAMP/PKA/CREB pathway: (A) qRT-PCR analysis of osteogenic transcription factors (Runx2 and Osterix) and marker genes (OPN and OCN), along with cell cycle gene (CyclinD1), after 7 days of osteogenic induction. Data normalized to GAPDH and expressed relative to control. (B, C) Western blot analysis and quantification of osteogenic proteins and CyclinD1 expression. Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Marker, Control, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: Immunohistochemical analysis confirms activation of osteogenicmarkers and the signaling pathway: (A) representative immunohistochemical staining and quantitative analysis of osteogenic transcription factors (Runx2 and Osterix), bone matrix proteins (OPN and OCN), and pathway molecules (p-PKA and p-CREB) in distraction gap tissues at 6 weeks of consolidation. Brown staining indicates positive expression. (B) CGRP treatment significantly enhanced all markers (scale bars: 50 μm). Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Immunohistochemical staining, Activation Assay, Staining, Expressing

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 of frequency ( B ) of GLI1 - / FMOD - , GLI1 + / FMOD - , GLI1 + / FMOD + , GLI1 - / FMOD + cells in the injured site of FOP model mice at 3 dpi ( n = 5 per group). P values from left to right: ** P = 2.27e-3, **** P = 1.17e-12, *** P = 4.59e-4, **** P = 6.94e-13, **** P = 2.49e-11. Scale bar, 200 or 20 μm. ( C, D ) Representative immunofluorescence images ( C ) and statistical analysis of frequency ( D ) of GLI1 - / SOX9 - , GLI1 + /SOX9 − , GLI1 − /SOX9 + , GLI1 + / SOX9 + cells in the injured site of FOP model mice at 5 dpi ( n = 5 per group). P values from left to right: * P = 2.52e-2, * P = 1.85e-2, **** P = 3.43e-5, *** P = 4.18e-4. Scale bar, 200 or 20 μm. ( E , F ) Representative immunofluorescence images ( E ) and statistical analysis of frequency ( F ) of GLI1 − /RUNX2 - , GLI1 + /RUNX2 − , GLI1 − /RUNX2 + , GLI1 + /RUNX2 + cells in the injured site of FOP model mice at 5 dpi ( n = 5 per group). P values from left to right: **** P = 5.56e-5, **** P = 1.32e-6, ** P = 1.12e-3, **** P = 1.73e-5. Scale bar, 200 or 20 μm. ( G , H ) Representative immunofluorescence images ( G ) and statistical analysis of frequency ( H ) of GLI1 - /COL2 - , GLI1 + / COL2 − , GLI1 + / COL2 + , GLI1 - / COL2 + cells in the injured site of FOP model mice at 7 dpi ( n = 5 per group). P values from left to right: **** P = 1.52e-10, **** P = 2.90e-11, **** P = 6.99e-11. Scale bar, 200 or 20 μm. ( I ) Representative immunofluorescence images showing Gli1 + tendon sheath progenitors differentiate into osteocytes at 14 dpi in FOP model mice. Scale bar, 200 or 20 μm. ( J ) Statistical analysis of the frequency of GLI1 - /OCN - , GLI1 + /OCN + , GLI1 - /OCN + , GLI1 + /OCN - cells in the injured site of FOP model mice at 14 dpi ( n = 5 per group). P values from left to right: **** P = 8.87e-11, **** P = 9.49e-7, **** P = 4.35e-10, **** P = 3.85e-6, **** P = 6.35e-5. ( K , L ) Representative Safranine O staining images ( K ) and statistical analysis ( L ) of chondrocyte region in the injured tendon of Gli1 - CreER T2 ; Acvr1 R206H/+ and Gli1 - CreER T2 ; Acvr1 R206H/+ ; DTA mice ( n = 5 per group). *** P = 3.27e-4. Scale bar, 200 μm. ( M , N ) Representative microCT images ( M ) and statistical analysis ( N ) of HO in the injured tendon of Gli1 - Cre ERT2 ; Acvr1 R206H/+ and Gli1 - Cre ERT2 ; Acvr1 R206H/+ ; DTA mice at 63 dpi ( n = 5 per group). **** P = 7.86e-7. Data are presented as mean ± SD. All P values were determined by One-way ANOVA with Bonferroni post hoc test ( B , D , F , H , J ) and unpaired Student’s t test ( L , N ).

Article Snippet: Anti-mouse OCN , Proteintech , Cat#23418-1-AP.

Techniques: Immunofluorescence, Staining