TCF4 and EGR1 self-regulation mediate EGR1 overexpression in ibrutinib-resistant cells. (A) The peaks of TCF4 ChIP-seq in HBL1 ABC DLBCL cells and ATAC-seq in the ibrutinib-resistant pool and parental HBL1 cells located at genomic regions of the EGR1 gene from a recent study.12 (B) Immunoblot analysis of TCF4 expression in ibrutinib-resistant cells of OCI-Ly10, TMD8, and Rec-1 compared with that in their parent cells. β-actin served as a loading control. (C) TCF4 ChIP-qPCR analysis with the indicated 5 pairs of primers for the EGR1 promoter and transcription start site regions. Immunoglobulin G (IgG) served as a control. Error bars represent mean ± SD (∗P < .05; n = 3). (D) TCF4 binds to the EGR1 promoter region (top) and induces EGR1 transcription by a standard dual luciferase reporter gene assay in 293T cells (bottom) (∗∗P < .01; ∗∗∗P < .001; n = 3). Firefly luciferase activity from the pGL3 basic reporter was normalized with β-galactosidase activity. (E) Immunoblot analysis of EGR1 and TCF4 expression after knockdown of TCF4 by 2 shRNAs or a control shRNA. β-Actin served as a loading control. (F) The peaks of EGR1 ChIP-seq OCI-Ly10 or TMD8 cells on genomic regions of the EGR1 gene from our recent study.14 EGR1 shRNA expression sample served as a ChIP control in OCI-Ly10 cell line. (G) EGR1 ChIP-qPCR analysis with the same EGR1 primers as in panel C. IgG served as a control. Error bars represent mean ± SD (∗∗∗∗P < .0001; n = 3). (H) EGR1 binds to its own promoter region for transcription. The same luciferase reporter gene constructs were used as in panel D (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; n = 3). Firefly luciferase activity from the pGL3 basic reporter was normalized with β-galactosidase activity.
