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ijc28116-sup-0001-suppfig1.TIF1831KFigure SI. Osteoblast identification and the effects of BDNF on osteoblast differentiation. (A) Human bone marrow mesenchymal stromal cells (MSCs) (2 x 103 cells/ml) in passage 3–5 were incubated in osteogenic differentiation medium (or aMEM containing 10% FCS as a control) for 21 days to obtain osteoblasts. Then, matrix mineralization was analyzed by alizarin red S staining, and the ALP level in cells was analyzed by alkaline phosphatase (ALP) staining. (B) Human MSCs in passage 3–5 were incubated in osteogenic differentiation medium in the absence or presence of BDNF (25ng/ml) and K252a to obtain osteoblasts. No significant differences of mineralization were found between ost group, ost+BDNF group, and ost+BDNF+K252a group. (C) Cell lysate was collected at day 7, 14, and 21 respectively. Then ALP activity was determined by colorimetric assay. No significant difference was detected between ost group, ost+BDNF group, and ost+BDNF+K252a group.
ijc28116-sup-0002-suppfig2.TIF1286KFigure S2. Coculture systems for studying the effects of BDNF on osteoclast formation and RANKL/OPG expression. (A) In the co-culture system of OBs and preOCs, OBs were cultured in the upper chamber of 12-well transwell inserts with 0.4-μm pores, and preOCs were planted in the lower chamber. (B) In the non-contacted co-culture system of HMCLs and OBs, HMCLs were cultured in the upper chamber of 12-well transwell inserts with 0.4-μm pores, while OBs were cultured in the lower chamber. (C) In the cell-to-cell contacted co-culture system of HMCLs and OBs, HMCLs were cultured in the upper chamber of 12-well transwell inserts with 1.0-μm pores, while OBs were cultured on the backside of the insert membrane, as previously described by Yaccoby S, et al. (D) RANKL mRNA levels in each group were detected by PCR. The direct contact of osteoblast and RPMI8226 markedly increased RANKL mRNA expression in osteoblasts (lane 5 compared with lane 3). However, these effects were significantly reversed by a neutralizing antibody to BDNF (lane 6 compared with lane 5).
ijc28116-sup-0003-suppfig3.TIF384KFigure S3. Down-regulation of protein expression confirmed by western blot analysis. (A) Osteoblasts (80–90% confluence) were transfected with TrkB-siRNA or the scrambled control siRNA. Western blot analysis confirmed that TrkB expression in osteoblasts was significantly downregulated by TrkB-siRNA (lane 3) compared to control-siRNA (lane 2). (B) RPMI8226 cells were transfected with BDNF antisense shRNA (AS-RPMI8226) or empty-vector shRNA (EV-RPMI8226) by replication-incompetent lentiviral vectors. Western blot analysis confirmed that both WT-RPMI8226 (lane 1) and EV-RPMI8226 cells (lane 2) expressed BDNF, and endogenous BDNF was knocked down in AS-RPMI8226 cells (lane 3) compared to EV- and WT-RPMI8226 cells.
ijc28116-sup-0004-suppinfo1.TIF538KSupporting Information
ijc28116-sup-0005-suppinfo2.TIF201KSupporting Information

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