MIP-1d Activates NFATc1 via PLCc2 and NFkB Figure 4. MIP-1d promotes osteoclastogenesis and bone resorption in vivo. Mice (5/group) were administered MIP-1d (1 ng/ml) or PBS subcutaneously over the calvariae twice each day for 5 days. Calvariae were removed and paraffin-embedded sections were generated for histomorphometric analysis. (A) OCL were identified by TRAP staining, counted under light microscopy (right, 400X), and reported relative to mm of marrow cavity surface (left). (B) Resorption area (left) was determined by measuring the total area occupied by marrow cavities within each calvarial section (right, 100X). Data are representative of two independent experiments, expressed as the mean (n = 10) 6 s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. **, p,0.01. doi:10.1371/journal.pone.0040799.g004 intervals. A minimum of three sections were stained with orange G and TRAP, and light microscopic images were acquired. The number of OCL/mm of marrow cavity surface and total resorption area (the total area occupied by marrow cavities) between the coronal and lamboid sutures was measured using MetaMorph image analysis software (Universal Imaging Corp., Sunnyvale, CA). Quantitative (q)RT-PCR Total RNA was extracted using Trizol (Invitrogen) and cDNA was generated by reverse transcription. 25 ml reactions contained 1X SYBR Green Reaction Mix (Applied Biosystems), 1 ml cDNA, and 100 nm of each primer: CTSK (sense) 59- CTCGGCGTTTAATTTGGGAGA-39, (antisense) 59- TCCAGGTTATGGGCAGAGATT-39; TRAP (sense) 59- GTGCTGCTGGGCCTACAAAT-39, (antisense) 59- TTCTGGCGATCTCTTTGGCAT-39; DC-STAMP (sense) 59- TGGAAGTTCACTTGAAACTACGTG-39 (anti-sense) 59- CTCGGTTTCCCGTCAGCCTCTCTC-39; GAPDH (sense) 59- AACTTTGGCATTGTGGAAGG-39, (antisense) 59-ACA- CATTGGGGGTAGGAACA-39. qRT-PCR parameters were: 1 cycle (95uC for 3 minutes) and 40 cycles (95uC for 30 seconds, 61.9uC for 30 seconds, and 72uC for 45 seconds). Amplification of GAPDH was used as an internal control. Relative expression between samples was calculated by the comparative CT method. Western and EMSA Analysis BMM were cultured in media without serum or M-CSF for 6 hours and subsequently treated with MIP-1d (0.1 pg/ml) for time periods indicated in Figs. 2, 3. For some experiments, BMM were cultured in the presence of chemical inhibitors specific to the NF- kB (IKK-2 inhibitor VIII, EMD Chemicals, Gibbstown, NJ) and PLCc2 (U73122, Cayman Chemical, Ann Arbor, MI) pathways as indicated in Fig. 3E,F. IKK-2 inhibitor VIII and U73122 were used at 20 mM and 5 mM, respectively, consistent with that utilized in similar studies [15,28,29]. Protein expression/phosphorylation was detected as described [12]. For examination of nuclear protein levels and EMSA analysis, nuclear cell lysates were generated using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Scientific, Rockford, IL). Antibodies were obtained from Cell Signaling (Danvers, MA) unless otherwise indicated: ERK, p- ERK, JNK, p-JNK, PLCc2, p-PLCc2, IkBa, p-IkBa, p50, p52, RelA, c-Rel, RelB, c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), and NFATc1 (Santa Cruz Biotechnology). b-actin (Sigma- Aldrich, St. Louis, MO) and HDAC1 served as loading controls. PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40799