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. 2021 Jul 27;40(37):5613–5625. doi: 10.1038/s41388-021-01961-9

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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. 8 — a siRNA mediated silencing of B-Myb and E2F2. Stable B-Myb overexpression (LV-B-Myb, upper panel) HCT116 cells were transiently transfected with negative control siRNA, B-Myb siRNA, or E2F2 siRNA. Stable B-Myb knockdown (shB-Myb, lower panel) HCT116 cells were transiently transfected with pcDNA3.0 empty vector, LV203-B-Myb-Flag and pcDNA3.0-E2F2 expression constructs. Twenty-four hours after transfection, qRT-PCR was performed to determine B-Myb and E2F2 expression. b E2F2 is required for B-Myb-induced cell proliferation. Cells were transiently transfected as described in (a), and cell proliferation was detected by CCK8 assay at the indicated time points. c GSEA plot showing that the B-Myb-regulated genes correlate with CSR_LATE gene signatures (CSR_LATE_UP.V1_UP). d B-Myb is essential to activation of ERK and AKT pathways. Immunoblotting was performed to determine B-Myb, ERK, p-ERK, and p-AKT expression in the stable B-Myb overexpression (LV-B-Myb) and its control (LV-vector) HCT116 cells (Left). Stable B-Myb knockdown (shB-Myb) and its control (shNC) HCT116 cells were subjected to serum starvation for 2 h, and then treated with EGF (200 ng/mL) for 15 min. Immunoblotting was performed to determine p-ERK expression (Right). e E2F2 is required for B-Myb-induced activation of ERK and AKT pathways. Stable B-Myb overexpression (LV-B-Myb) HCT116 and RKO cells were transiently transfected with negative control (NC) siRNA, B-Myb siRNA and E2F2 siRNA, respectively. Forty-eight hours later, cells were subjected to immunoblotting with the indicated antibodies. The band intensity ratios of pAKT or pERK to GAPDH are shown below the bands. f B-Myb and E2F2 activate ERK and AKT pathways. Stable B-Myb knockdown (shB-Myb) HCT116 and RKO cells were transfected with pcDNA3.0 empty vector, LV203-B-Myb-Flag and pcDNA3.0-E2F2 expression constructs, respectively. Forty-eight hours later, cells were subjected to immunoblotting with the indicated antibodies. g Work model. B-Myb and E2F2 regulate the transcription of each other (reciprocal feed-forward regulation) as well as their own transcription (autoregulation). In addition, B-Myb and E2F2 associate with each other (collaboration) and regulate various downstream target genes to activate ERK and AKT pathways and induce the malignant cell phenotype in colorectal cancer. Data represent the mean ± SD. All experiments were performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001.