Fig. 1.

IKKβ^SSEE induces bona fide osteoclasts from bone marrow, spleen, and fetal liver progenitors. (A) Bone marrow macrophages from IKKβ knockout and littermate mice were infected with retroviruses expressing GFP, IKKβ^WT, or IKKβ^SSEE and cultured with M-CSF alone or M-CSF + RANKL for 4 days (as described under “Materials and Methods”) and were TRACP stained to visualize osteoclasts. Arrows denote osteoclasts. Lower panels, quantification; flx = loxP flanked. For bone resorption, fetal liver cells (FLCs) were plated on dentin slices to determine bone resorption. After 5 days, slices were stained with toluidine (Tol.) blue and phalloidin to visualize resorption tracks (darker areas) and actin ring formation, respectively. Cells were cultured in M-CSF alone except where indicated. Quantification of percent resorbed area was done using Bioquant. Percent resorption area of dentin slices for each condition is denoted. (B) Expression levels of IKKβ^WT and IKKβ^SSEE in control and IKKβ knockout macrophages were measured by Western blot using anti-IKKβ antibody. Expression of endogenous NEMO is shown as control. (C) Spleen cells were plated as detailed under “Materials and Methods.” After 3 days, cells were infected with the various pMx viruses as indicated. Cells expressing the viral proteins were selected with puromycin for 2 to 3 days. Western blot for expression of NF-κB molecules and osteoclast markers in total cell lysates of spleen cells infected with the indicated viruses was performed using equal amounts of total proteins. Actin expression indicates equal loading. OC+ = osteoclast positive control total cell lysate. (B, C) pMx = retroviral expression vector. (D) Relative expression of mRNA for osteoclast markers by real-time qPCR. mRNA was extracted from cells infected with the indicated viruses. Relative expression of the indicated osteoclast marker genes was determined using specific primers outlined under “Materials and Methods.” GAPDH served as internal standard for cDNA normalization. Values are expressed as relative quantity plus standard error of the mean. (E) Control and IKKβ knockout monocytes were transduced with viruses expressing GFP or the indicated forms of IKKβ [active (SSEE) and inactive (KM, SSAA) forms]. These cells were treated with M-CSF and RANKL and TRACP stained. (F) Western blot to demonstrate expression of the indicated IKKβ constructs. Parallel cells treated as shown in panel E were lysed and subjected to Western blots with IKK and actin antibodies.