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. 2023 Jun 22;14:3729. doi: 10.1038/s41467-023-39256-0

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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. 9 — a Schematic illustration showing the procedures for the generation of two stable, TMX-inducible MEF cell lines (Nat10^{+/Cre, lox/lox}) from the Ubc-cre; Nat10^lox/lox embryos following a 3T3 protocol (see the “Methods” section). Inducible Nat10 KO was achieved by 2 µM OHT treatment for three consecutive days. Rescue of Nat10 was attained by overexpression (OE) of WT Nat10 ORF plasmid. b Western blot displaying the NAT10 and PCNA protein levels in WT and OHT (Ubc-cre; Nat10^lox/lox) MEF line. α-TUBULIN was used as a loading control. c Quantitative RT–PCR results showing the relative expression levels of Nat10 in WT and OHT MEF line. Data are presented as mean ± SEM, n = 3. ****p < 0.0001 by two-tailed Student’s t-test. d Cell cycle analysis by flow cytometry between WT and OHT MEF cells. G1, p = 0.0083; S, p = 0.0017; G2/M, p < 0.0001; p-value are analyzed by two-tailed Student’s t-test. e Annexin V apoptosis detection of WT and OHT MEF cells. Q4, Viable cells, p = 0.2983; Q3, Early apoptosis, p = 0.2147; Q2, Late apoptosis, p = 0.3122; Q1, Necrotic cells, p = 0.7863. p-value are analyzed by two-tailed Student’s t-test. f CCK8 assay showing the cell proliferation rates among WT, OHT treatment and OHT plus Nat10 overexpression (OE) groups. Data are presented as the mean ± SEM, n = 3 biologically independent samples and analyzed by two-tailed Student’s t-test. g, h Comparison of cell proliferation as visualized by Ki67 labeling in g and quantification in h among WT (empty vector), OHT treatment and Nat10 overexpression groups. Scale bar, 50 μm. Data are presented as mean ± SEM, n = 3. *p < 0.05, **p < 0.01 by two-tailed Student’s t-test. Vector-OHT, p = 0.0026; OHT-Nat10, p = 0.0340. i, j The polysome profiling displaying the translational efficiency and ribosome assembly in MEF cells analyzed by sucrose density gradient centrifugation. The graphic curves showed the polysome profiles of MEF cells treated by mock (Blue) or OHT (Red) in i. Comparison of the ratios of 60S to 40S (Left) and of 80S to 40S (Right) in MEF cells with mock treatment (Blue) or OHT (Red) in (j). Data are presented as mean ± SEM, n = 3. ***p < 0.001 by two-tailed Student’s t-test. 60S/40S, p = 0.0008; 80S/40S, p = 0.0003. n = 3 biologically independent samples were included in each group (b, d, e, g, i). Source data are provided as a source data file.