Journal: Advanced Science

Article Title: Neuronal Induction of Bone‐Fat Imbalance through Osteocyte Neuropeptide Y

doi: 10.1002/advs.202100808

Figure Lengend Snippet: Osteocyte NPY‐induced BMSC fate switching is mediated by the inhibition of TEAD1 and JUNB through cAMP/PKA/CREB signaling. qRT‐PCR analysis of Tead1 and Junb expression in BMSCs receiving different treatments under A) osteogenic or B) adipogenic induction for 24 h, or C) in undifferentiated BMSCs with different treatments for 24 h. n = 3 per group. D) qRT‐PCR and E) western blotting confirmed the overexpression efficiency of recombinant Tead1 and Junb lentiviruses in BMSCs. n = 3 per group. F) ARS and ORO staining images and G) quantification of the positively stained areas showing the reduced activity of NPY to inhibit osteogenesis and promote adipogenesis of BMSCs overexpressing Tead1 or Junb . Scale bar: 50 µm. n = 3 per group. ELISA for cAMP in BMSCs receiving different treatments under H) osteogenic or I) adipogenic induction for 24 h. n = 3 per group. PKA activity assay for cell homogenates of BMSCs receiving different treatments under J) osteogenic or K) adipogenic induction for 24 h. n = 3 per group. L) Western blotting for CREB and phosphorylated CREB (p‐CREB) in BMSCs receiving different treatments under osteogenic or adipogenic induction for 24 h. qRT‐PCR analysis of M) Tead1 and N) Junb expression in BMSCs receiving different treatments under osteogenic induction for 24 h. n = 3 per group. O) ARS and ORO staining images and P) quantification of the positively stained areas in BMSCs receiving different treatments under osteogenic or adipogenic induction. Scale bar: 50 µm. n = 3 per group. Q) ChIP‐qRT‐PCR analysis shows enrichment of the CREB antibody (CREB Ab)‐immunoprecipitated Tead1 or Junb promoter region relative to the input DNA. Normal rabbit anti‐IgG served as a negative control. Histone H3 antibody (Histone H3 Ab) pulldown for the enrichment of Rpl30 gene served as a positive control. n = 3 per group. Data are presented as mean ± SD. For panels (A), (B), (G), (H–K), (M), (N), and (P): unpaired, two‐tailed student's t ‐test (differences between un‐induced and control groups in (A), (B), and (H–K), or between control and NPY groups in (G), (M), (N), and (P)) or one‐way ANOVA combined with Bonferroni post hoc test (differences among other groups except un‐induced group in (A), (B), and (H–K), or except control group in (M), (N), and (P)). For panel (C) and (D): one‐way ANOVA combined with Bonferroni post hoc test. For panel (Q): unpaired, two‐tailed student's t ‐test (differences between control and NPY groups for Rpl30 ) or two‐way ANOVA combined with Bonferroni post hoc test (differences among other groups for Tead1 or Junb ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: 24 h later, the culture medium was replaced with fresh osteogenic or adipogenic medium (Cyagen) supplemented with OCY‐CM (300 μg mL −1 at the protein level) from different groups, recombinant NPY protein (0.1 n m ; MedChemExpress), BIBO3304 (0.1 n m ; Selleck Chemicals, Houston, USA), NPY (0.1 n m ) + db‐cAMP (10 μ m ; MedChemExpress), NPY (0.1 n m ) + H‐89 (10 μ m ; MedChemExpress), NE (0.1 n m ; Sigma‐Aldrich), ACh (0.1 n m ; Sigma‐Aldrich), or an equal volume of vehicle.

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Recombinant, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Negative Control, Positive Control, Two Tailed Test, Control