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. 2022 Apr 11;5:343. doi: 10.1038/s42003-022-03250-x

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© The Author(s) 2022

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 LEVER and ε-globin expression after LEVER knock-out (KO) in K562 cells measured by RT-qPCR. LEVER RNA expression is measured by primers detecting four selected regions (marked in Fig. 1d) in non-targeting control (NTC-gRNA) or LEVER knock-out (LEVER-gRNA) K562 cells. Data from n = 3 biological replicates are shown. Error bars represent SEM. Statistical difference was calculated using Welch’s one-tailed t-test. b Chromatin Immunoprecipitation qPCR (ChIP-qPCR) of RNA Pol II at ε-globin and γ-globin promoters in non-targeting control (NTC-gRNA) and LEVER knock-down (LEVER KD) cells. ACTB and MYT1 serve as Pol II ChIP positive and negative controls, respectively. Pol II occupation at ε-globin promoter is measured by two primer sets (ε-globin P1 and ε-globin P2). Data from technical replicates of one biological sample is shown. c ε-globin protein abundance in LEVER KO or KD K562 cells measured by western blot. β-actin serves as the loading control. d LEVER and ε-globin expression in inducible LEVER KD K562 cells with (+) or without (−) doxycycline treatment for the indicated duration. LEVER expression is quantified at the LVR-4 region. Data from n = 3 biological replicates are shown. Error bars represent SEM. Statistical difference was calculated using Welch’s one-tailed t-test comparing with the −7d sample. ns, not significant. e Time course RT-qPCR analysis of LEVER RNA and ε-globin in K562 cells during 7-day hypoxic culture under 1% oxygen. Data from technical replicates of one biological sample is shown. f ε-globin protein abundance in K562 cells cultured under 1% oxygen. α-tubulin serves as the loading control.