Fig. 6: IBSP and miR-19a cooperatively induce osteoclastogenesis and promote cancer cell growth in the bone. | Nature Communications

Fig. 6: IBSP and miR-19a cooperatively induce osteoclastogenesis and promote cancer cell growth in the bone.

From: Exosomal miR-19a and IBSP cooperate to induce osteolytic bone metastasis of estrogen receptor-positive breast cancer

Fig. 6

a, b MCF7 cells were injected into the tibias of mice. These mice also received 2 µg exosomes prepared from MCF7BoM2 and 0.5 µg recombinant IBSP injected into the tibias every 2 days. The exosomes prepared from MCF7BoM2/miR-19aKO and PBS were used as controls. After three weeks, the bioluminescence signals of tibial bones were measured. Two-sided student’s t-tests were performed. p = 0.4670 (Exo19aKO + PBS vs Exo19aKO + IBSP, n = 4 vs 6), p = 0.1949 (Exo19aKO + PBS vs Exo + PBS, n = 4 vs 6), p = 0.0446 (Exo19aKO + PBS vs Exo + IBSP, n = 4 vs 6). Data are presented as mean values ± SEM (a). The bones were then removed and bone densities were measured by X-ray. Two-sided student’s t-tests were performed; p = 0.9280 (Exo19aKO + PBS vs Exo19aKO + IBSP, n = 4 vs 6), p = 0.9143 (Exo19aKO + PBS vs Exo + PBS, n = 4 vs 6), p = 0.0257 (Exo19aKO + PBS vs Exo + IBSP, n = 4 vs 6) Data are presented as mean values ± SEM (b). c The removed bones were decalcified, sectioned, and examined for the amount of OC surface by TRAP staining followed by quantification using the ImageJ. Two-sided student’s t-tests were performed; p = 0.7224 (Exo19aKO + PBS vs Exo19aKO + IBSP, n = 4 vs 6), p = 0.8773 (Exo19aKO + PBS vs Exo + PBS, n = 4 vs 6), p = 0.0002 (Exo19aKO + PBS vs Exo + IBSP, n = 4 vs 6). Data are presented as mean values ± SEM. d Integrin αVβ3 was seeded on the bottoms of 96-well plates. IBSP was then added to the well in the presence or absence of the library of natural compounds. The bound IBSP was measured by colorimetric substrates of alkaline phosphatase (AP). The OD 405 was compared between CGA-treated groups and the DMSO-treated group. Two-sided student’s t-tests were performed; p < 0.0001 (n = 3) in all groups. CGA was found to most significantly inhibit the binding of IBSP to integrin αVβ3. Data are presented as mean values ± SEM. e The role of IBSP as a chemoattractant of osteoclast cells, and the inhibitory effect of CGA on this chemoattraction were examined by the µ-Slide Chemotaxis assay. The movement of 20 OC cells with the highest mobility was tracked and the directionality was calculated by Chemotaxis and Migration Tool. p-value was calculated with the Rayleigh test (left: p = 0.0000002, right: p = 0.194763).

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