Journal: Stem Cell Research & Therapy
Article Title: Depletion of mmu-miR-185 enhances osteocyte connectivity and suppresses bone fragility through the interaction between ARL8B and RAB5A
doi: 10.1186/s13287-025-04612-y
Figure Lengend Snippet: Depletion of miR-185 in MLO-Y4 cells reduces the osteoclastogenic potential of RAW264.7 cells. ( A ) The levels of soluble RANKL (sRANKL) and osteoprotegerin (OPG) in the serum of WT and miR-185 KO mice were quantified by ELISA ( n = 3). ( B ) The concentrations of sRANKL and OPG in conditioned medium from WT and miR-185 KO MLO-Y4 cells were measured by ELISA ( n = 3). ( C ) RAW264.7 cells were co-cultured for 4 days with conditioned medium derived from miR-185 KO, sh Arl8b , and corresponding control MLO-Y4 cells, supplemented with 50 ng/mL RANKL and 30 ng/mL M-CSF ( n = 3). After 4 days of culture, qPCR was performed to evaluate mRNA expression levels of osteoclast marker genes, including Trap , DC-stamp , Ctsk , and Nfatc1 . ( D ) Representative images of tartrate-resistant acid phosphatase (TRAP)-stained osteoclast cultures ( n = 3). ( E ) TRAP-positive cells were counted based on the number of nuclei. ( F ) TRAP-positive multinucleated cells (containing > 3 nuclei) were quantified. ( G ) Cathepsin K (CTSK), a marker of osteoclasts, was detected by western blotting ( n = 3). ( H ) The mRNA expression of matrix metalloproteinase 14 ( Mmp14 ) in MLO-Y4 cells was measured by RT-qPCR ( n = 3). ( I , J ) The protein levels of MMP14 in MLO-Y4 cells were evaluated by western blotting. ( K ) Quantitative analysis of MMP14 protein expression was performed using ImageJ software. ( L )The mRNA expression of Mmp14 in the femurs of mice was assessed by qPCR ( n = 3). Data represent at least three independent experiments and are shown as mean ± S.D. (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001). The full-length blot is presented in supplementary Raw Western
Article Snippet: RANKL and OPG protein levels in the supernatants were determined using the sRANKL ELISA Kit (CUSABIO, cat# CSB-E05127m) and the OPG ELISA Kit (CUSABIO, cat# CSB-E04693m), respectively.
Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Control, Expressing, Marker, Staining, Western Blot, Quantitative RT-PCR, Software