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. 2021 Mar 17;12:1714. doi: 10.1038/s41467-021-21976-w

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, b Staining of TRAP (a) and TRAP occurrence rates at the bone-tumor interface of bone samples bearing DU 145 (b, left), PC-3 (b, middle), and C4-2B (b, right) cancer cells with different acetylation statuses of KLF5. Black arrows in panel a indicate TRAP occurrence at the interface of bone and tumor areas (marked by B and T respectively). Scale bar in a, 100 μm. For DU 145 tibial tumors, EV, KLF5, KLF5^KR (KR), and KLF5^KQ (KQ) had 3, 5, 4, and 8 tumors respectively. For PC-3 tibial tumors, each group had six tumors. For C4-2B cells, n = 8 tumors for each group. c, d Differentiation of preosteoclast RAW264.7 cells into TRAP-positive osteoclasts after co-culturing with DU 145 (d, left), PC-3 (d, middle), and C4-2B (d, right) cells with different statuses of KLF5 acetylation, as indicated by TRAP staining (c, TRAP + multinucleated osteoclasts are marked by black arrows) and statistical analyses of TRAP + osteoclasts per well (d). n = 3 wells for each group in DU 145 and PC-3 cells. n = 4 wells for each group in C4-2B cells. e Expression of markers for osteoclast differentiation, including Trap, Nfatc1, Itgb3, c-Myc, Tm7sf4, Ctsk, Mmp9, and c-Src, by real-time qPCR using mouse-specific primers in the co-cultures of RAW264.7 mouse pre-osteoclasts and DU 145 cells with different forms of KLF5. f, g Staining of TRAP (f) and statistical analysis of TRAP + osteoclasts per well (g) in RAW264.7 pre-osteoclasts treated with CM from DU 145 cells with different forms of KLF5 for 6 days. Black arrows in f indicate TRAP + multinucleated osteoclast cells, and n = 3 wells for each group in g. h Detection of osteoclast differentiation markers in CM-treated RAW264.7 pre-osteoclasts by real-time qPCR. For both co-culture and CM treatment experiments, Rankl (20 ng/ml) was added to maintain a basal level of osteoclastogenesis. Scale bars in c and f, 50 μm. In panels b, d and g, data are shown in mean ± S.E.M. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t test). Source data are provided as a Source Data file.