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. 2023 Feb 1;42(12):911–925. doi: 10.1038/s41388-023-02602-z

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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. 2 — A RT-qPCR analysis of METTL3 mRNA level (upper) in the non-tumorigenic mammary epithelial cell line MCF10-A and the breast cancer cell lines MCF7 and MDA-MB-231. METTL3 is normalized to β-ACTIN. Western blot of METTL3 (lower) on whole cell extracts (WCE) from MCF10-A, MCF7 and MDA-MB-231 cell lines. β-ACTIN (ACTB) is used as the loading control. B LC-MS/MS quantification of m⁶A, m¹A and m⁶A_m in mRNA of MCF10-A, MCF7 and MDA-MB-231 cell lines. Methylated adenosines are normalized to the total of unmodified adenosines. RT-qPCR analysis and western blot of METTL3 mRNA and protein levels, respectively in METTL3 knockdown (sh1 and sh2) and scramble (scr) control in MCF10-A (C), MCF7 (D) and MDA-MB-231 (E) cell lines. METTL3 is normalized to β-ACTIN. Cell proliferation rate of scramble (scr) and METTL3 knockdowns (sh1 and sh2) in MCF10-A (F), MCF7 (G) and MDA-MB-231 (H) assessed over 4 days. Colony formation assay in MCF10-A (I), MCF7 (J) and MDA-MB-231 (K) cells in scramble (scr) and upon depletion of METTL3 (sh1 and sh2) at 7 days after seeding. Quantification of the relative number of colonies was calculated with scr set as 1. Percentage of apoptotic cells in control and METTL3 depleted cells in MCF10-A (L), MCF7 (M), and MDA-MB-231 (N) cell lines. + indicates Annexin V positive cells; and – indicates Annexin V negative cells. Volcano plots representing the Log₂ fold change of differentially expressed genes upon METTL3 knockdown (sh1 and sh2) in MCF10-A (O), MCF7 (P) and MDA-MB-231 (Q) cell lines in comparison to control cells (scr). The significant up- and down-regulated genes are shown in red and blue, respectively. P-value < 0.05. Data are mean ± SEM; n = 3; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.