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. 2019 Jun 26;10:2795. doi: 10.1038/s41467-019-10677-0

Fig. 3.

Fig. 3

Age-related TGFβ activation promotes TRAF3 degradation. a TRAF3 and GAPDH WBs of tibial metaphyses, femoral BM and cortical bone from 3- and 18-m-old C57BL/6 mice. b Densitometry of TRAF3 WBs of bones from patients. Mean ± SD; 8–18-years (n = 26), 53–59-years (n = 11), 60–69-years (n = 10), 70–87-years (n = 8 biologically independent samples; *p < 0.05, **p < 0.01). c TRAF3 and osteocalcin (Ocn) immunostained vertebral sections. TRAF3+/Ocn+ osteoblasts (yellow arrows) on trabecular bone (Tr.B) surfaces and TRAF3+ hematopoietic cells in BM. Mean ± SD (n = 4 biologically independent samples; **p < 0.01). Scale bar, 50 μm. d TRAF3 and GAPDH WBs of BdMPCs treated with PBS, TNF (20 ng/ml), TGFβ1 (1 ng/ml), BMP2 (100 ng/ml), or PTH (80 ng/ml) for 8 h. e WB of TGFβ1 in tibial metaphyseal lysates. f Active TGFβ1 levels in serum and BM from 2.5- and 19-m-old C57BL/6 mice. Mean ± SD (n = 7 biologically independent samples; **p < 0.01). g Total and active TGFβ1 levels in vertebral lysates. Mean ± SD (children (8–18-years) n = 22; and adults (55–87-years) n = 20 biologically independent samples; *p < 0.05). h BdMPCs treated with vehicle or TGFβ1+/−chloroquine (100 μM) or MG132 (20 μM) for 8 h. IP using anti-Ub Ab and WB with TRAF3 Ab. Likely mono- and poly-ubiquitinated TRAF3 (lower and upper arrowheads, respectively). i Calvarial pre-OBs treated with vehicle or TGFβ1+/−300  nM AT406 for 8 h. IP with anti-TGFβRI Ab and WB with cIAP1/2, TRAF3 and TGFβRI Abs, or IP with anti-cIAP1/2 Ab and WB with TRAF3 Ab. j WB of cIAP2, TRAF3, and GAPDH in cells in (i). k Areas of ALP+ cells from BdMPCs treated with TGFβ1+/−AT406 for 5 days. Mean ± SD (n = 4 biologically independent samples; *p < 0.05, **p < 0.01). l IF and area of TRAF3 and LAMP2 co-localization in BdMPCs treated with vehicle or TGFβ1 plus chloroquine for 8 h. Mean ± SD (n = 4 biologically independent samples; **p < 0.01). Scale bar, 20 μm. m WB of TRAF3 and GAPDH in BdMPCs treated with vehicle or TGFβ1+/−chloroquine for 8 h. n Areas of ALP+ cells from WT and cKO BdMPCs treated with vehicle or TGFβ1+/−chloroquine for 5 days. Mean ± SD (n = 5 biologically independent samples; *p < 0.05, **p < 0.01 vs. TGFβ1 alone. Analyses in (c, g, l) done using unpaired Student's t test and in (b, f, k, n) using one-way ANOVA with Tukey’s post-hoc test). All in vitro experiments repeated twice with similar results. Tβ1: TGFβ1 (1 ng/ml)