Assessing the importance of interferon-stimulated gene upregulation in the action of the IMiDs. (A) IFIT3 expression (mean RPKM ± SD) from RNA sequencing in OPM2 cell after deletion of Ikaros, Aiolos, or lenalidomide (Len) 10 μM; and MM1S and H929 cells after deletion of Ikaros, vs control. Data are described in Figure 3. (B) Western blot OPM2 and H929 clones of IKZF1 20 (IK20) gRNA (2 clones) and an EV control, performed 4 days after gRNA induction with dox. Membranes were probed for Ikaros, IFIT3, and GAPDH as loading control. (C) Analysis of Ikaros binding in the human B-cell line GM12878 by ChIP-seq from the ENCODE Project. Shown is the mapping track of sequence reads at the IFIT3 locus. Note the presence of 2 alternative first IFIT3 exons. Ikaros conservative called peaks are shown as bars in the top panel. (D) Time course of cell viability assayed by flow cytometry using a fixable viability dye. Relative viability vs non-dox-treated control (set to 100%) is shown. Data are representative of 2 experiments. Graph shows live cell numbers in H929 cells expressing the control EV or IKZF1 gRNA (IK20) after dox gRNA induction (day 0), Len 1 μM treatment (day 1), and/or a titration of β-IFN (day 2). IU, international units. (E) CellTiter Glo viability assay in H929, MM1S, OPM2, and U266 cells 3 days posttreatment with Len 1 μM and/or β-IFN 500 IU. Graph depicts the mean luminescent reading from the Cell Titer Glo assay expressed as an arbitrary unit (AU) ± SD for each sample from 3 experiments.