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. 2020 Mar 12;11:1344. doi: 10.1038/s41467-020-15126-x

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© The Author(s) 2020

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 Western blot analysis of Mettl16 expression from ceca of CONV, GF, ex-GF, abx, vanco, Am, and Lp mice. Actin served as loading control. The membrane and thus the actin loading control for the blot displaying samples from ex-GF, Am, and Lp mice is identical to the western blot in Supplementary Fig. 4a. b Quantification using six different western blots; CONV n = 20, ex-GF n = 6; GF n = 13; abx n = 9; vanco n = 8; Am n = 9; Lp n = 9; Mettl16/Actin ratio was normalized to ratio in CONV mice in order to compare protein expression across multiple Western Blots. Ordinary one-way ANOVA was performed. *p-value < 0.05; p-values (compared to CONV): ex-GF: 0.9518; GF: 0.0255; abx: 0.0431; vanco: 0.1750; Am: 0.1783; Lp: 0.2728) (Holm–Sidak’s multiple comparisons test). c Representation of two m⁶A peaks (mean of read per million normalized coverage (RPM) in detected methylation peaks) from anti-m⁶A immunoprecipitates and input in the 3′UTR of the Mat2a transcript in cecum. The peaks designated a and b were visualized for the indicated mouse models using IGV; d Quantification of indicated Mat2a peaks (a, b from b) as -log2 normalized read counts. Ordinary one-way ANOVA for multiple comparisons was performed. CONV (n = 15), GF (n = 12); ex-GF (n = 4); abx (n = 9); vanco (n = 8); Am (n = 11), Lp (n = 3); two independent sequencing experiments; a p-values (all compared to CONV): ex-GF: 0.6458; GF: 0.0039; abx: 0.0572; vanco: 0.6458; Am: 0.6458; Lp: 0.4695; b p-values (all compared to CONV): ex-GF: 0.889955; GF: 0.000323; abx: 0.003604; vanco: 0.889955; Am: 0.00000000000028; Lp: 0.018424; e anti-m⁶A-IP and qRT for Mat2a transcript in CONV (n = 11), ex-GF(n = 5) and GF (n = 8) cecal RNA; IgG-IP (n = 4) served as control for unspecific binding; ordinary one-way ANOVA was performed; *p-value < 0.05; ***p-value < 0.005; p-values (compared to CONV): ex-GF: 0.6897; GF: 0.0127; IgG: 0.0029; f Western Blot analysis of Mat2a expression in CONV, GF, ex-GF, abx, vanco, Am, and Lp cecum. Actin served as loading control. Asterisk marks an unspecific band. g Quantification of Mat2a expression in CONV, GF, ex-GF, abx, vanco, Am, and Lp cecum. Quantification was performed using at least three different western blots for each condition; CONV n = 15, ex-GF n = 6; GF n = 12; abx n = 12; vanco n = 12; Am n = 9; Lp n = 9; Mat2a/Actin ratio was normalized to ratio in CONV mice in order to compare protein expression across multiple western blots; ordinary one-way ANOVA was performed. ***<0.0001, **p-value < 0.005 (Holm–Sidak’s multiple comparisons test); p-values (all compared to CONV): ex-GF: 0.157; GF: 0.00000019; abx: 0.0002; vanco: 0.0019; Am: 0.1111; Lp: 0.154; data are presented as mean values ±SEM throughout the figure; details for statistical analysis and original data for a, b, d–g are given in the source data file.