MIP-1d Activates NFATc1 via PLCc2 and NFkB Figure 1. MIP-1d enhances RANKL-induced osteoclastogenesis in vitro. BMM or RAW 264.7 cells were treated with MIP-1d (0.1 pg/ml) and RANKL (5 ng/ml) individually or in combination for 7 days. (A) OCL formation was assessed by counting the total number of OCL per well under light microscopy (D, 200X Magnification; Scale bars, 50 mm). (B, C) Fusion index was determined by counting the number of nuclei per OCL in 15–20 random fields and is reported as (B) the average number of nuclei per OCL and (C) the number of OCL containing a specified range of nuclei per cell (BMM shown, similar results were observed for RAW 264.7). Data are representative of three independent experiments performed in quadruplicate, expressed as the mean (n = 12) 6 s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. **, p,0.01; ***, p,0.001. doi:10.1371/journal.pone.0040799.g001 MIP-1d Enhances Osteoclastogenesis and Bone Resorption in vivo Although MIP-1d levels are elevated in several conditions causing pathological bone loss [12,24] and we have demonstrated its ability to enhance OCL differentiation in vitro, the capacity of MIP-1d to induce osteolysis in vivo has not been investigated. To determine whether MIP-1d affects osteoclastogenesis and bone resorption in vivo, we utilized a murine model wherein MIP-1d or PBS was injected subcutaneously over the calvariae. Consistent with the ability of MIP-1d to enhance OCL differentiation in vitro, calvariae from mice administered MIP-1d showed a significant increase in OCL number and resorption area relative to that observed in mice administered PBS (Fig. 4). These data indicate that MIP-1d is able to increase osteoclastogenesis and bone PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40799