Table 1.
Sequences of primers used for gene cloning and quantification of gene expression by quantitative real-time PCR.
Fig 1.
Overexpression of METRNL suppresses AP-1 transcription factor complex activity in 293T cells.
In cells co-transfected with pCDNA3.1-METRNL, pAP-1, and pRL-TK, METRNL suppressed basal endogenous AP-1 transcription factor complex activity. METRNL also inhibited AP-1 transcription factor complex activation in cells treated with exogenous AP-1 activity stimulator (PMA and ionomycin). Data showing means from three independent experiments are presented after normalization for Renilla luciferase activity. *P < 0.01
Fig 2.
Immunohistochemistry for METRNL protein in rat bone.
(A) In western blotting analyses, METRNL expression is observed in lysates (L) and supernatants (S) of stable CHO cells overexpressing His-tagged METRNL, indicating that METRNL indeed is a secreted protein and the polyclonal antibody was specific to METRNL (N for lysates of CHO cells). (B) METRNL is detected in hypertrophic chondrocytes (→) in the tibial growth plate of three-week-old rats. (C) In the primary spongiosa, METRNL is detected in osteoblasts (→) lining the trabecular bone surfaces, but osteocytes (▼) are absent.(D) In the secondary spongiosa, METRNL protein is detected weakly and diffusely in osteoblasts lining the bone surfaces (→).
Fig 3.
Stable overexpression of METRNL may inhibit MG63 cell differentiation.
(A) METRNL-EGFP fusion protein was highly expressed in the cytoplasm of MG63 cells. (B) Real-time PCR data suggest that METRNL mRNA levels in the METRNL overexpression group were significantly higher than those in the control. (C) Overexpression of METRNL suppresses mineralized nodule formation in MG63. Mineralized nodule formation is observed in EGFP-overexpressing cells in mineralizing medium. Mineralized nodule formation is absent in cells overexpressing pEGFP-METRNL. (D, E) Quantification of OCN and OPG, detected by RT-PCR after treatment with mineralization condition medium for 7 days and 14 days, revealed remarkable downregulation of these genes in the METRNL overexpression group compared with controls (*P < 0.05).