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. 2021 Mar 19;12(4):297. doi: 10.1038/s41419-021-03566-2

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© The Author(s) 2021

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 Volcano plot of differentially expressed genes after circSPI1 knockdown. The vertical lines correspond to 2.0-fold up and down, and the horizontal line represents a P value of 0.05. B The expression changes of representative DEGs after circSPI1 knockdown were validated by RT-qPCR. C Representative DEGs upon circSPI1 knockdown were essential for the survival of AML cells. Normalized read counts of sgRNA in AML cells transfected with the CRISPR-cas9/sgRNA library before and after population doublings were showed. D Functional enrichment analysis showing significant GO terms and hallmark gene sets among circSPI1-regulated genes. E GSEA showing that differentiation and apoptosis were enriched using shcircSPI1-regulated genes. F The overlap of regulated genes between shcircSPI1 and shSPI1. G Top ten enriched functional terms for shared genes between circSPI1 and SPI1. H Differentiation was enriched using SPI1-regulated genes. I Heatmap showing that myeloid differentiation-associated genes were oppositely regulated by circSPI1 and SPI1. J The protein level of SPI1 was detected with or without circSPI1 knockdown. GAPDH served as the loading control. The original gels and three replicates are presented in Supplementary Fig. 3A. K CircSPI1 interacted with eIF4AIII in AML cells. The binding potential of circSPI1 with eIF4AIII was predicted by the Interactome database (upper panel). The interaction between circSPI1 and eIF4AIII was detected by western blotting combined with RNA pulldown (lower panel). Total lysates prepared from NB4 cells were subjected to pulldown with circSPI1 and negative control probes, followed by western blotting. The original gels and three replicates are presented in Supplementary Fig. 3B. L Functional enrichment of genes regulated by circSPI1 but not SPI1 was summarized using annotated GO terms and hallmark gene sets. All bar graphs represent the average of three independent replicates and error bars are SD, *P < 0.05, **P < 0.01, ***P < 0.001.