Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Osteoblasts respond to PTH treatment with increased Slit3 transcription and translation. mRNA expression of the Slit3 in the endplate tissue of WT young mice treated with Veh and aged mice treated with Veh or PTH for 1 month ( a ), and WT LSI mice treated with veh or PTH for 2 months ( b ) ( n = 3, one-way ANOVA with Tukey’s multiple comparisons test). c mRNA expression of the Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and subsequently treated with Veh or PTH at different doses for 3 days ( n = 3, t -test). d , e Protein expression level of Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and treated with Veh (PBS) or PTH for 3 days. ( n = 3, t -test). Representative images ( f ) and quantification ( g ) of IF staining of PGP9.5 positive primary DRG neuron fibers crossing the microgroove barrier (microfluid assay) cultured in condition medium (CM), CM + PTH, CM + PTH plus mouse Slit3 antibody (1 µg/mL), or CM + Veh (1X PBS) plus human recombinant Slit3 (1.25 µg/mL) for 1 week. Representative images showing co-immunostaining for OCN (green) and Slit3 (red) in the lumbar spine sections and quantitative analysis of number of Slit3 + cells in trabecular bone (TB) and endplate (EP) of aged mice ( h – j ) and PPR OCN −/− LSI mice ( k – m ) treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: MC3T3 subclone 4 cell line was purchased from ATCC (CRL-2593 TM ) and cultured using Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate, but without ascorbic acid (A10490-01, Thermo Fisher).

Techniques: Expressing, Cell Culture, Staining, Recombinant, Immunostaining

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Transcriptional mechanism underpinning Slit3 secretion. a mRNA expression levels of Ets1, E47, FoxJ2 , and FoxA2 genes in MC3T3 cells cultured in osteoblast differentiation-inducing medium and treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, t -test). b , c Protein expression level of E47, FoxA2 in MC3T3 cells cultured in stimulated medium and treated with PTH or Veh (PBS) for 3 days ( n = 3, t -test). Representative images depicting E47 IF staining (green) in lumbar vertebral body and endplate sections ( d ), quantitative analysis of the number of E47 + cells in the vertebral body ( e ) and endplate ( f ) of aged mice treated with Veh or PTH ( n = 5, t -tset). Scale bar: 100 µm. Representative images showing FoxA2 IF staining (red) in lumbar vertebral body and endplate sections ( g ), quantitative analysis of the number of FoxA2 + cells in the vertebral body ( h ) and endplate ( i ) of aged mice treated with PTH or vehicle for 2 months ( n = 5, t -tset). Scale bar: 100 µm. j Relative fold enrichment of the E47 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). k Relative fold enrichment of the FoxA2 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: MC3T3 subclone 4 cell line was purchased from ATCC (CRL-2593 TM ) and cultured using Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate, but without ascorbic acid (A10490-01, Thermo Fisher).

Techniques: Expressing, Cell Culture, Staining, Binding Assay

Journal: Scientific Reports

Article Title: Understanding of the different roles of Noggin in the Noggin-BMP-2 and Noggin-BMP-9 dimer complexes at the molecular level

doi: 10.1038/s41598-025-33735-8

Figure Lengend Snippet: Initial and final structures of Noggin-BMP-2 and Noggin-BMP-9 after 100 ns MD simulation. Identification of the binding effect of BMP-2 and BMP-9 to BMPR2 in MC3T3-E1 Cells by Noggin using Immunoprecipitation. ( A ) Initial structures of Noggin-BMP-2 and ( B ) Initial structures of Noggin-BMP-9 ( C ) Aligned structures of initial (White color) and final structures of Noggin-BMP-2 (Coral/Blue) ( D )Aligned structures of initial (White) and final structures of Noggin-BMP-9 (Green/Ultraviolet) ( E ) RMSD of Noggin-BMP-2 and Noggin-BMP-9 complexes during the all-atom MD simulation (100 ns). ( F ) RMSF (Root Mean Squared Fluctuation) of Noggin-BMP-2 and Noggin-BMP-9 complexes during the all-atom MD simulation (100 ns). RMSD and RMSF values ​​represent the mean and standard deviation of three independent simulations. ( G ) Western blot analysis using total cell lysates. ( H ) Immunoprecipitation (IP) of BMPR2 followed by immunoblotting (IB) to detect BMP-2 and BMP-9 binding.

Article Snippet: The mouse pre-osteoblastic cell line MC3T3-E1 was purchased from the American Type Culture Collection (ATCC, CRL-2593, Manassas, VA, USA).

Techniques: Binding Assay, Immunoprecipitation, Standard Deviation, Western Blot

Journal: Scientific Reports

Article Title: Understanding of the different roles of Noggin in the Noggin-BMP-2 and Noggin-BMP-9 dimer complexes at the molecular level

doi: 10.1038/s41598-025-33735-8

Figure Lengend Snippet: Identification of binding effect of BMP-2 and BMP-9 to BMPR2 in MC3T3-E1 cells by Noggin. ( A ) BMP-2–treated group, ( B ) BMP-2 + Noggin–treated group, ( C ) BMP-9–treated group, ( D ) BMP-9 + Noggin–treated group. DAPI (blue), anti-BMPR2 (red), and anti-BMP-2 or anti-BMP-9 (green). Merged images show co-localization (yellow). Scale bar: 10 μm; magnification: ×600. ( E ) Western blot of SMAD1/5/9 phosphorylation under the indicated treatments. Top, p-SMAD1/5/9; middle, total SMAD1/5/9; bottom, GAPDH. ( F) Alkaline phosphatase (ALP) activity measured 60 min after BMP addition using a p-nitrophenyl phosphate colorimetric assay (405 nm). Bars indicate mean ± SD ( n ≥ 3). p < 0.0001 vs. NT for all treated groups; asterisks denote significance relative to NT.

Article Snippet: The mouse pre-osteoblastic cell line MC3T3-E1 was purchased from the American Type Culture Collection (ATCC, CRL-2593, Manassas, VA, USA).

Techniques: Binding Assay, Western Blot, Phospho-proteomics, Activity Assay, Colorimetric Assay