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. 2023 Nov 13;14(11):738. doi: 10.1038/s41419-023-06263-4

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© The Author(s) 2023

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. 3 — First, METTL14 was transfected into RAW264.7 cells and prepared for further study. A TRAP staining and F-actin band staining were applied to detect osteoclast differentiation between the two groups. Scale bar: 200 μm. B Histograms of the number, coverage rate and nuclei of TRAP-positive osteoclasts between the two groups. C Relative expression levels of Ctsk, MMP9 and Acp5 in RAW264.7 cells between the two groups. After ZOL stimulation, si-METTL14 was transfected into RAW264.7 cells and prepared for further study. D TRAP staining and F-actin band staining were applied to detect osteoclast differentiation between the two groups. Scale bar: 200 μm. E Histograms of the number, coverage rate and nuclei of TRAP-positive osteoclasts between the two groups. F Relative expression levels of Ctsk, MMP9 and Acp5 in RAW264.7 cells between the two groups. G Heatmap showing the differentially expressed genes of osteoclast differentiation after ZOL stimulation. H KEGG analysis revealing the differentially expressed genes from three intersections: osteoclast differentiation genes, MeRIP hyper genes and mRNA down genes. I RT-qPCR analysis revealing the expression levels of 10 differentially expressed genes after ZOL stimulation or METTL14 overexpression. J Schematic diagram showing the genomic location of NFATc1. K m6A peak visualisation of meRIP-Seq in NFATc1 transcripts with or without ZOL stimulation. The m6A peaks are in the 3’UTRs of NFATc1. L The potential m6A methylation loci of NFATc1 on the SRAMP website. M We divided 9 segments of NFATc1 according to the potential m6A methylation loci. N m6A-RT-qPCR assay was performed to detect the enrichment of 9 segments of NFATc1 between the anti-m6A group and anti-IgG group after ZOL stimulation. O An m6A-RT-qPCR assay was performed to detect the enrichment of the NFATc1–9, 10 segment as the ZOL concentration increased. P An m6A-RT-qPCR assay was performed to detect the enrichment of the NFATc1–9, 10 segment under ZOL or si-METTL14 stimulation. Q, R Two m6A modification sites were validated through step-by-step mutation of luciferase reporters. Data are representative of three independent experiments expressed as the mean ± SD (*p < 0.05).