Figure 1.
Figure 1. CRISPR-Cas9 deletion of IKZF1/3 recapitulates the action of the IMiDs in MM cells. (A) Depiction of full-length IKZF1 gene and CRISPR targeting strategy. (B) Western blot of Cas9 expressing OPM2, MM1S, and H929 clones of IKZF1 gRNA IK20 or an empty vector (EV) control performed 4 days after gRNA induction with doxycycline (dox). Membranes were probed for Ikaros, Aiolos, IRF4, MYC, and GAPDH as a loading control. (C) CellTiter Glo (CTG) viability assay 6 days after dox treatment of cells (as in panel B). Relative cell viability vs non-dox-treated control (set to 100%) is shown. Data are the mean ± SD from 3 experiments. (D) Depiction of full-length IKZF3 gene and CRISPR targeting strategy. (E) Western blot of Cas9-expressing OPM2 clones of each IKZF3 gRNA (Ai35 and Ai38) vs EV 4 days after dox treatment. Membranes were probed for Aiolos, Ikaros, IRF4, MYC, and GAPDH as a loading control. (F) CTG viability assay 6 days after dox treatment of OPM2 clones (as in panel E). Relative cell viability vs non-dox-treated control (set to 100%) is shown. Data are the mean ± SD from 3 experiments. (G) Western blot of OPM2 cells 2 days posttreatment with lenalidomide (Len, 1 or 10 μM) vs control, probed for Ikaros, Aiolos, MYC, IRF4, and GAPDH as a loading control. (H) Western blot of Cas9 expressing OPM2 cells (lanes 1 and 2) or IKZF3 gRNA Ai35-1 clone (lanes 3 and 4), transduced with IKZF1 IK20 gRNA (lanes 2 and 4) or EV control (lane 1) and bulk flow cytometry sorted for mCherry (Cas9), GFP (IK20 gRNA) expression. Analysis was performed 72 hours after dox treatment to induce gRNA expression. Membrane were probed for Ikaros, Aiolos, IRF4, and GAPDH as a loading control. (I) Flow cytometry viability time course (using a fixable viability dye and counting beads to quantify live cells) after dox treatment in OPM2 IK20 (bulk), Ai35-1 (clone), combination IK20 + Ai35-1, or EV control cells (as in panel H). Relative cell viability vs non-dox-treated control (set to 100%) is shown. Data are the mean ± SD from 3 experiments. (B,E,G,H) Molecular weights (MW) are indicated to the right of the plots. ****P < .0001, using an unpaired Student t test.

CRISPR-Cas9 deletion of IKZF1/3 recapitulates the action of the IMiDs in MM cells. (A) Depiction of full-length IKZF1 gene and CRISPR targeting strategy. (B) Western blot of Cas9 expressing OPM2, MM1S, and H929 clones of IKZF1 gRNA IK20 or an empty vector (EV) control performed 4 days after gRNA induction with doxycycline (dox). Membranes were probed for Ikaros, Aiolos, IRF4, MYC, and GAPDH as a loading control. (C) CellTiter Glo (CTG) viability assay 6 days after dox treatment of cells (as in panel B). Relative cell viability vs non-dox-treated control (set to 100%) is shown. Data are the mean ± SD from 3 experiments. (D) Depiction of full-length IKZF3 gene and CRISPR targeting strategy. (E) Western blot of Cas9-expressing OPM2 clones of each IKZF3 gRNA (Ai35 and Ai38) vs EV 4 days after dox treatment. Membranes were probed for Aiolos, Ikaros, IRF4, MYC, and GAPDH as a loading control. (F) CTG viability assay 6 days after dox treatment of OPM2 clones (as in panel E). Relative cell viability vs non-dox-treated control (set to 100%) is shown. Data are the mean ± SD from 3 experiments. (G) Western blot of OPM2 cells 2 days posttreatment with lenalidomide (Len, 1 or 10 μM) vs control, probed for Ikaros, Aiolos, MYC, IRF4, and GAPDH as a loading control. (H) Western blot of Cas9 expressing OPM2 cells (lanes 1 and 2) or IKZF3 gRNA Ai35-1 clone (lanes 3 and 4), transduced with IKZF1 IK20 gRNA (lanes 2 and 4) or EV control (lane 1) and bulk flow cytometry sorted for mCherry (Cas9), GFP (IK20 gRNA) expression. Analysis was performed 72 hours after dox treatment to induce gRNA expression. Membrane were probed for Ikaros, Aiolos, IRF4, and GAPDH as a loading control. (I) Flow cytometry viability time course (using a fixable viability dye and counting beads to quantify live cells) after dox treatment in OPM2 IK20 (bulk), Ai35-1 (clone), combination IK20 + Ai35-1, or EV control cells (as in panel H). Relative cell viability vs non-dox-treated control (set to 100%) is shown. Data are the mean ± SD from 3 experiments. (B,E,G,H) Molecular weights (MW) are indicated to the right of the plots. ****P < .0001, using an unpaired Student t test.

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