PIM2 expression is transcriptionally regulated by IRF4 in MM cells. (A) Whole cell lysates, which were extracted from the MM cell lines, RPMI 8226, MM.1S, U266, NCI-H929, KMS-11, and INA-6, primary CD138-positive cells derived from patients with MM, and PBMCs from healthy donors were subjected to immunoblotting. β-Actin served as a loading control. (B) Publicly available ChIP-seq data (GSM1195560) were analyzed for the distribution of IRF4 binding at the PIM2 locus in MM.1S cells. The x-axis shows the genomic position. KK1, which is an adult T-cell leukemia/lymphoma cell line, was used as a negative control (GSM2481669). (C) RPMI 8226 cells were transduced with shLuc or shIRF4 (#1). Transduced cells were subjected to a ChIP-Q-PCR analysis for IRF4 occupancy on the PIM2 gene. Results were normalized to control IgG. Error bars show the SD of triplicates. ∗∗P < .01 significantly different from the shLuc condition; the Student t test. (D) Total RNA was extracted from RPMI 8226 cells transduced with shLuc or shIRF4 (#1, #2) and then subjected to Q-PCR. GAPDH served as an internal control. Values represent the amount of mRNA relative to the shLuc control. Error bars show the SD of triplicates. ∗∗∗P < .001 significantly different from the control; the Tukey-Kramer multiple comparison test. (E) RPMI 8226, MM.1S, and U266 cells were transduced with shLuc or shIRF4 (#1, #2). The whole cell lysates extracted were subjected to immunoblotting using the indicated antibodies. β-Actin served as a loading control. Relative expression levels of each target, which are normalized to its loading control, are shown below for each immunoblotting image. PBMCs, peripheral blood mononuclear cells.