Fig. 5.

The effect of rhSCL on endogenous levels of MEPE-ASARM. NHBCs were cultured under mineralizing conditions for 35 days in chamber slides and then treated for a further 3 days in the absence or presence of rhSCL (50 ng/mL). Cells were stained by immunofluorescence antibodies for (A) MEPE-ASARM and (B) PHEX and analyzed by confocal microscopy. Representative images of triplicate wells are shown for each treatment. The relative mean fluorescence intensity ± SEM (arbitrary units) of the cell layer for each treatment was quantified using ImageJ analysis of images (n =6) taken of the unmerged antibody-specific (FITC) signal. (C) The level of background staining using a normal rabbit IgG as the primary antibody. Nuclei were stained using DAPI, as described in “Materials and Methods.” (D) The effect of a 3-day treatment of day 35 differentiated NHBC cultures with rhSCL (0 to 50 ng/mL) on full-length MEPE expression assessed by Western blot. Relative MEPE levels were compared with those of β-actin, which served as a loading control, and represent four independent blots. In all cases, an asterisk indicates difference from untreated (p <.05).