Effects of 5TGM1-GFP-TK MM cells on MC-4 cell expression of OB markers, transcription factors, and mineralization. MC-4 cells were cocultured with either empty transwells or transwells containing 5TGM1-GFP-TK (5TGM1) cells for either (A) 12 hours or (B) 3, 6, and 12 hours before removing the transwells and isolating RNA from the MC-4 cells for quantitative PCR analysis of Bsp, Ocn, Runx2, and Osx expression, as indicated, relative to expression in the absence of 5TGM1-GFP-TK. (C) The MC-4 cells (2 × 105/60 mm dish) were cultured in α-MEM plus ascorbic acid for 12 days (the media was changed every 2 days), and 5TGM1 cells (2 × 106) were added at 5 days to half the wells. The 5TGM1 cells were removed before the MC-4 cells were stained with von Kossa for mineralization and were photographed using a light box with no magnification. (D) MC-4 cells (1 × 105/well 24-well plate) were pretreated with either 0.01 μg of IgG or neutralizing antibodies to IL-7 and TNF-α for 2 hours and then cocultured with 5TGM1 cells in a transwell for 4 hours, followed by RNA isolation from the MC-4 cells and quantitative PCR analysis of Runx2 expression relative to MC-4 cells cultured with an empty transwell. Higher doses of the antibodies did not have additional effects (data not shown). (E) MC-4 cells were treated with the indicated concentrations of IL-7 and TNF-α (when both added, amount indicated is for each) for 6 hours, and Runx2 expression was analyzed by quantitative PCR.
