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Fig. 3 Effects of EN on Dex-induced zebrafish larvae bone loss. (a) Zebrafish larvae were treated with EN (0.2, 1, and 5 ?M) combined with 10 ?M Dex, and skull mineralization ability was determined using ARS. (b) Mineralization area. (c) Mineralization IOD. (d) The mRNA expression of osteoblast-related genes (ALP, RUNX2a, SP7, and OCN) on 9-dpf in Dex-induced zebrafish larvae. (e) The mRNA expression of osteoclast-related genes (CTSK, RANKL, NFATC1, and TRAF6) on 9-dpf in Dex-induced zebrafish larvae. The data are shown as the means ± SD (n = 13 zebrafish larvae/group). ###P < 0.001 vs. Con; ?P < 0.05, ??P < 0.01, ???P < 0.001 vs. Dex. Scale bar: 100 ?m. EN, eurycomanone; Dex, dexamethasone; ARS, alizarin red S; IOD, integrated optical density; ALP, alkaline phosphatase, RUNX2a, runt-related transcription factor 2a; SP7, Sp7 transcription factor; OCN, osteocalcin; CTSK, cathepsin K; RANKL, receptor of nuclear factor-?B; NFATC1, nuclear factor of activated T cells 1; TRAF, TNF receptor-associated factor 6; dpf, days after fertilization; Con, control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)