Fig 2: RANKL expression is increased in TRAF3 cKO osteoblastic cells. a Calvarial pre-OBs and spleen cells isolated from 7-day-old WT or cKO pups, co-cultured with 10−8 M 1,25(OH)2Vitamin D3 −/+ RANK:Fc (1 µg/ml) for 7 days and TRAP-stained. b Osteoclast numbers per well (Oc.N/well) in (a) were counted. Mean ± SD (n = 4 biologically independent samples; #p < 0.05, **p < 0.01). c WB of TRAF3, OPG, RANKL, and GAPDH in protein lysates from tibial metaphyseal bone from 3- and 9-m-old WT and cKO mice. d Densitometry analysis of RANKL/OPG protein ratio in (c). Mean ± SD (n = 3 biologically independent samples; *p < 0.05). e RANKL mRNA expression in BdMPCs from 3- and 9-m-old WT and cKO mice. Mean ± SD (n = 3 biologically independent samples; *p < 0.05, **p < 0.01). f BdMPCs from 3-m-old WT and cKO mice treated with TGFβ1 (1 ng/ml) for 0, 0.5, and 8 h. WB of RelA, RelB, HDAC2, and GAPDH in nuclei and cytoplasm. g Scheme for mouse RANKL promoter analysis showing putative κB binding sites and primer design to test binding sites. h, i Sheared chromatin from WT and cKO BdMPCs was used to perform DNA IP using h RelA, i RelB Abs or IgG control. Real-time PCR performed using designed primers that contain the putative κB binding sites 1, 2, 3, or an unrelated site, normalized to the input. Mean ± SD; n = 3 biologically independent samples; *p < 0.05. j Sheared chromatin from WT (W) and cKO (K) BdMPCs used to perform DNA IP using RelA, RelB, or IgG control Abs. PCR performed using designed primers that contain the putative κB binding sites 1, 2, 3, and an un-related site. All analyses done using one-way ANOVA with Tukey’s post-hoc test. All the in vitro experiments repeated twice with similar results

Fig 3: RelA and RelB increase RANKL expression by MPCs. Calvarial pre-osteoblasts from 7-day-old WT mice were infected with GFP control, RelA, or RelB retroviruses for 48 h. a RelA and b RelB mRNA expression tested by real-time PCR to confirm successful over-expression. Mean ± SD (n = 3 biologically independent samples; **p < 0.01; unpaired Student's t test). c RANKL mRNA expression tested by real-time PCR. Mean ± SD (n = 3; *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s post-hoc test). d Culture media collected from culture wells and RANKL protein levels measured by ELISA. Mean ± SD (n = 3 biologically independent samples; no significant difference; one-way ANOVA with Tukey’s post-hoc test). e Membrane-bound RANKL levels in 50,000 CD45-Sca-1+ MPCs tested by flow cytometry and expressed as mean fluorescence intensity (MFI). Average of 2 biologically independent samples from two individual experiments. All the in vitro experiments were repeated twice with similar results