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. 2022 Aug 11;41:243. doi: 10.1186/s13046-022-02447-6

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — HIF-1α stabilizes C/EBP-β LAP mRNA, increasing ABCB1 and ABCC1, and decreasing ABCA1. NCI-H2228 cells were cultured as indicated in Fig. 1a. a Immunoblot of C/EBP-β LAP in whole cell extracts. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. b Normoxic NCI-H2228 cells, wild-type (wt), overexpressing C/EBP-β LAP (LAP⁺) or overexpressing C/EBP-β LIP (LIP⁺), were lysed and immunoprecipitated with an anti-C/EBP-β antibody, recognizing both LAP and LIP isoforms, then immunoblotted for HIF-1α. An aliquot of the lysates before the immunoprecipitation was directly probed with the anti-HIF-1α antibody, to check that the protein was equally present. The image is representative of 1 out of 3 experiments. No Ab: wild-type cells subjected to immunoprecipitation without the anti-C/EBP-β antibody, as negative control. c RNA-IP with an anti-HIF-1α antibody, followed by RT-PCR amplification (in technical triplicates) with primers for C/EBP-β LAP (upper panel) or LIP (lower panel) isoforms. Data are means±SD (n = 4 biological replicates). **p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells. d ChIP of C/EBP-β on ABCB1, ABCC1 and ABCA1 promoter, in technical triplicates. Data are means±SD (n = 4 biological replicates). *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells. e-h Hypoxic (H) NCI-H228 cells were treated with a non-targeting sequence (scr) or with two shRNAs (sh1, sh2) targeting HIF-1α. e HIF-1α mRNA level, measured by RT-PCR in technical triplicates. Data are means±SD (n = 4 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells. f Immunoblot of HIF-1α. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. g RNA-IP with an anti-HIF-1α antibody, followed by RT-PCR amplification (in technical triplicates) with primers for C/EBP-β LAP. Data are means±SD (n = 4 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: sh1/sh2-cells versus scr-cells. h ChIP of C/EBP-β on ABCB1, ABCC1 and ABCA1 promoter, in technical triplicates. Data are means±SD (n = 4 biological replicates). *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells, °°°p < 0.001: sh1/sh2-cells versus scr-cells