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CSF-1 Regulation of Osteoclastogenesis for Tooth Eruption

¹ Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA; and
² Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA;

View larger version (44K): [in a new window]   Figure 1. OPG gene expression in the dental follicle of the first mandibular molar, showing the comparison of normals (N) vs. tl/tl mutants at different post-natal days. Comparisons are within a given day, because pups at different days are not littermates. For a given day, 3–4 pairs of littermates were analyzed. (A) Agarose gel picture from electrophoresis of semi-quantitative RT-PCR. (B) OPG gene expression ratio from semi-quantitative RT-PCR as normalized to their actin controls. (C) Real-time PCR showing gene expression of the mutants relative to that of their littermate controls, which was set as 1. Note that at all days except day 5, OPG gene expression is higher in the tl/tl mutants, and that for days 3, 7, and 11, this expression is significantly different (*) from the controls at P 0.05 by Student’s t test. Relative gene expression was calculated by the Delta CT method, which was normalized to the endogenous control of ß-actin. Error bars in both B and C represent standard deviations (SDs).

View larger version (36K): [in a new window]   Figure 2. CSF-1 gene expression in dental follicle cells transfected with siRNA targeting CSF-1. Results in each panel are the mean of 3 separate experiments and were compared statistically by analysis of variance and separation of means, with least significant difference at P 0.05. (Panel A) siRNA concentration effect on reduction of gene expression of CSF-l after 48 hrs of transfection. Maximum inhibition of expression is seen with 80 nM CSF-1 siRNA. (Panel B) Time-course effect of siRNA on suppression of CSF-1 gene expression, showing suppression at all times, with maximal suppression at 72 hrs. Error bars in both panels represent SDs.

View larger version (65K): [in a new window]   Figure 3. Immunostaining of CSF-1 in dental follicle cells after 48 hrs of transfection with 80 nM of siRNA targeting the CSF-1 gene. Note that CSF-1 staining was reduced after siRNA treatment (B) as compared with the control (A). Controls for immunostaining in which the primary antibody was omitted did not stain (C).

View larger version (37K): [in a new window]   Figure 4. OPG expression in dental follicle cells transfected with CSF-1 targeting siRNA. Result in Panel B is the mean of 3 separate experiments. (Panel A) Semi-quantitative RT-PCR showing greater OPG expression after 48 and 72 hrs of transfection, in comparison with the controls at those times. (Panel B) Real-time RT-PCR results showing relative gene expression with controls (0 nM) set to 1 at each time point indicated. At both 48 and 72 hrs, the OPG gene expression is significantly increased (*) after transfection with 80 nM of CSF-1 siRNA (P 0.05), by Student’s t test. Relative gene expression was calculated by the Delta CT method, which was normalized to the endogenous control of ß-actin. Error bars represent SDs.