MIP-1d Activates NFATc1 via PLCc2 and NFkB Figure 3. MIP-1d induces expression and activity of c-fos and NFATc1 via stimulation of the PLCc2 and NF-kB signaling pathways. BMM were treated with or without MIP-1d (0.1 pg/ml). (A) c-fos and NFATc1 protein expression were determined by Western analysis performed on equal amounts of protein from total cell lysates. (B) Activation of AP-1 was determined 30 minutes following treatment with MIP-1d by non- radioactive EMSA analysis of nuclear cell lysates using AP-1 oligonucleotides. Oct-1 served as a loading control and 200-fold excess unlabeled oligonucleotide (200X-U.O.) served as a competitive control. (C) NFATc1 protein expression was determined 60 minutes following treatment with MIP-1d by Western analysis performed on equal amounts of protein from nuclear cell lysates. (D) CTSK, TRAP, and DC-STAMP mRNA expression were determined by qRT-PCR 24 hours following treatment with MIP-1d. Data are representative of three independent experiments performed in triplicate, expressed as the mean (n = 9) 6 s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. p,0.01; ***, p,0.001. (E, F) BMM were treated with MIP-1d (0.1 pg/ml) alone or in combination with the (E) NF-kB inhibitor IKK2 inhibitor VIII (IKK2-I) or the (F) PLC inhibitor U73122 and protein expression was determined 60 minutes (c-fos, NFATc1) or 30 minutes (IkBa, phospho-PLCc2) following MIP-1d treatment by Western analysis performed on equal amounts of protein from total cell lysates. (E) Densitometry values are provided below respective lanes for ease of interpretation. Data are representative of at least two independent experiments. doi:10.1371/journal.pone.0040799.g003 (P/S) using a 27 gauge needle. Cells were washed and re- suspended in complete media (aMEM +10% FBS +1% P/S) containing 5 ng/ml M-CSF (R&D Systems, Minneapolis, MN) and incubated in a 100 mm dish overnight. Non-adherent cells were then collected, strained through 70 mm nylon mesh, and treated with 50 ng/ml M-CSF for 3 days to generate M-CSF- dependent bone marrow macrophages (BMM). Osteoclast Differentiation Assay BMM were cultured in complete media containing M-CSF (20 ng/ml) and RANKL (5 ng/ml) (R&D Systems) in the presence or absence of full-length recombinant human MIP-1d (0.1 pg/ml) (R&D Systems). 50% of the culture media was replaced with fresh complete media containing cytokines as appropriate every 3 days. After 7 days, the development of mature osteoclasts (OCL) was determined by positive tartrate-resistant acid phosphatase (TRAP) staining (bright red color) and the presence of $3 nuclei under light microscopy. To determine the fusion index, the number of nuclei per OCL was counted in 15 to 20 random fields at 100X magnification. In vivo Calvarial Bone Resorption Following a previously established procedure [27], recombinant human MIP-1d (1 ng) or PBS was injected in a volume of 50 ml into the subcutaneous tissue overlying the right side of the parietal bones of calvariae in female 6-week-old CD-1 mice twice each day for 5 days. Four days following the last injection, calvariae were harvested, fixed in formalin for 24 hours, and decalcified in 10% EDTA, pH 7.4 for 48 hours. Paraffin-embedded sections (5 mm) were generated laterally from the sagittal suture at 100 mm PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40799