GCK is required for RAS^MutMM cells survival. (A) MM.1S (K-RAS^G12A), RPMI-8266 (K-RAS^G12A), and H929 (N-RAS^G13D) MM cells were infected by pLKO-Tet-On scramble control (shCNTL) or shGCK lentivirus and selected by puromycin (3 ug/μL) for 1 week. Knockdown of GCK by doxycycline (Dox) treatment (400 ng/mL) for 3 days was confirmed by western blotting. (B-D) Transduced and selected MM.1S, RPMI-8266, and H929 cells were cultured in the presence Dox (400 ng/mL) for 5 days. Cell proliferation was detected by (B) MTS assay, and cells were stained with PI for (C) cell-cycle analysis, or (D) with Annexin V and 7-AAD for apoptosis analysis. (E) U266 and LP-1 RAS^WT cells were infected by pLKO-Tet-On scramble control (shCNTL) or shGCK lentivirus and selected by puromycin (3 μg/μL) for 1 week. Knockdown of GCK by Dox treatment (400 ng/mL) for 3 days was confirmed by western blotting. (F-G) Transduced and selected U266 and LP-1 RAS^WT cells were cultured in the presence of Dox (400 ng/mL) to induce shRNA for 5 days. (F) Cell proliferation was detected by MTS assay, and (G) cells were stained with Annexin V and 7-AAD for apoptosis analysis.