Fig 2: IBSP-siRNA inhibited proliferation and induced apoptosis in CRC cells. B: blank, NC: negative control, *p < 0.05, **p < 0.01. (A) Downregulation of IBSP mRNA in CRC cells. (B) IBSP-siRNA inhibited cell proliferation of CRC cells. (C) IBSP-siRNA increased apoptosis of CRC cells. (D) The protein level of PCNA, Bcl2/Bax, Cyclin D1, and Cdk4 in CRC cells decreased after transfection with IBSP-siRNA

Fig 3: Ectopic expression of SPRY4 improves osteogenic differentiation of AIS MSCs in vitro.a, b AIS BM-MSCs from three patients were transduced with lentivirus overexpressing SPRY4 (lenti-SPRY4) or empty vector (lenti-NC). Efficiency of ectopic expression was verified by qRT-PCR (a) and western blot (b). c qRT-PCR analysis of osteogenic transcription factors and marker genes RUNX2, ALP, IBSP, OPN and COL1A1 on day 6 of osteogenic differentiation. d Western blot detected osteogenic transcription factors and marker genes IBSP, ALP, RUNX2 and OPN on day 6 of osteogenic differentiation. e, f ALP staining and relative ALP activity assays were performed on day 6 of osteogenic differentiation. g, h Calcium deposition by Alizarin red S staining and quantification were performed on day 12 of osteogenic differentiation. Data were from three independent experiments using BM-MSCs derived from three AIS patients. GAPDH was used as a loading control in both qRT-PCR and western blot analyses. Data are shown as the means ± SD

Fig 4: EphB4, TNFR2 and MAPK signaling pathways comprise a signaling axis to mediate the positive effect of TNF-a on osteogenic differentiation. a Levels of p38, p-p38, ERK1/2, p-ERK1/2, JNK1 + 2 + 3 and p-JNK1 + 2 + 3 in MC3T3-E1 cells treated with TNF-a for 0 min, 5 min, 15 min, 30 min and 60 min. b Levels of p38, p-p38, ERK1/2, p-ERK1/2, JNK1 + 2 + 3 and p-JNK1 + 2 + 3 in the pHBLV-TNFR2siRNA1 cells and the pHBLV-NC cells treated with or without 0.5 ng/ml TNF-a in regular culture medium for 15 min. c MC3T3-E1 cells were pretreated with or without 200 nM NVP-BHG712 in the regular culture medium for 1 h, and then 0.5 ng/ml TNF-a was added into the medium. The cells were incubated for another 15 min. Levels of ERK1/2 and p-ERK1/2 were determined. d-f MC3T3-E1 cells were cultured in the regular culture medium and pretreated with the ERK inhibitor U0126 (10 µM) for 1 h. The culture medium was then switched to the osteogenic induction medium supplemented with 0.5 ng/ml TNF-a and U0126 (10 µM). Cells treated without U0126 (10 µM) served as controls. ALP activities were determined 7d or 14d after the treatment (d). mRNA levels (e) and protein levels (f) of BSP and RUNX2 were determined 3 days after the treatment. *, p < 0.05 vs. the control group; **, p < 0.01 vs. the control group

Fig 5: Prostate cancer PC3 cell-derived HOXA11-AS modulates IBSP and CCL2 expression in SaOS2 osteoblastic cells in a paracrine manner. (A) The expression levels of well-known, cancer metastasis-associated lncRNAs (MALAT-1, NEAT-1, HOTAIR, and HOXA11-AS) in exosomes of prostate cancer cell lines by comparison of exosomal RNA versus total RNA. A lncRNA array dataset from NCBI Gene Expression Omnibus (GSE81034) was used; values represent raw signal intensities. (B) HOXA11-AS expression of PC3 stable cells overexpressing HOXA11-AS (designated as A11AS-PC3) and wild type PC3 cells (Wild); HOXA11-AS detection in conditioned medium (CM) of A11AS-PC3 and Wild. (C) HOXA11-AS expression of SaOS2 cells treated for 48 h with CM of A11AS-PC3 and Wild. (D) CCL2 and IBSP expression of SaOS2 cells treated for 48 h with CM of A11AS-PC3 and Wild. qPCR analyses were performed in triplicate (n = 3). Expression levels are presented relative to values of GAPDH, used as a reference gene, except for the right panel of B. The expression level of HOXA11-AS in CM of Wild was set to 1.0 in the right panel of B. Values represent mean ± standard deviation. Statistical significance was determined by unpaired t-tests. (E) Analysis of the size distribution of isolated EVs from wild type PC3-CM by the NanoSight nanoparticle tracking system (left panel). Comparison of relative abundances of HOXA11-AS in PC3 cell, and EV and EV-subtracted supernatant [EV-sup] fractions of PC3-CM by qPCR; equal amounts of RNA were applied (center panel). Relative abundances of HOXA11-AS in PC3 cell, and EV and EV-sup fractions are also shown as values per cell and values per mL of CM, respectively (right panel). The expression levels of PC3 were set to 1.0. Data are presented as mean ± standard deviation of triplicate data (n = 1).