EGR1 expression is elevated and further upregulated upon ibrutinib treatment in ibrutinib-resistant cells. (A) RNA-seq analysis shows significantly increased EGR1 expression in ibrutinib-resistant cell clones (IBR; 10 clones) compared with that in the parental (Par) cells (3 replicates). (B) Immunoblot analysis shows increased EGR1 protein levels in IBR clones. (C) IBR clones are not sensitive to BTK shRNA (left) or ibrutinib (right). SUDHL2 and OCI-Ly3 are ibrutinib-resistant cell lines used as controls. Error bars represent mean ± standard deviation (SD) (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; n = 3). (D) Immunoblot analysis shows increased EGR1 protein levels in IBR clones. (E) Immunoblot analysis shows increased EGR1 expression in resistant cells but reduced EGR1 expression in parental cells after 72 hours of ibrutinib treatment. (F) Immunoblot analysis shows increased EGR1 protein levels after 72 hours of ibrutinib treatment in all 4 patient samples. Protein bands were quantified by densitometry (∗P < .05; ∗∗P < .01). β-actin served as a loading control for all immunoblot experiments. DMSO, dimethyl sulfoxide; FPKM, fragments per kilobase of transcript per million mapped reads.