Knock-down of GCK decreases c-MYC, IKZF1, IKZF3, and BCL6 expression in RAS^MutMM cells. (A-C) MM.1S (K-RAS^G12D), RPMI-8266 (K-RAS^G12A), H929 (N-RAS^G13D), and U266 (RAS^WT) MM cells were infected by pPLKO-Tet-On scramble control (Tet-On-shCNTL) or sh-GCK lentivirus (Tet-On-shGCK), and selected by puromycin (3 μg/μL) for 1 week. To induce the shRNA, selected cells were treated with Dox 400 ng/mL for 3 days, and analyzed for GCK, IKZF1, IKZF3, c-MYC, and BCL-6 expression by western blotting. (A,B) β-actin was detected as loading control. For mRNA expression of GCK, IKZF1, and c-MYC by quantitative real-time PCR. Data were analyzed according to the ΔΔCt method. Results are shown as mRNA expression relative to control. (C) mRNA levels were normalized with β-actin mRNA expression as control. (D) Tet-On-shCNTL or Tet-On-shGCK MM.1S or (E) LP-1 cells were treated with Dox 400 ng/mL for 36 hours, starved in RPMI-1640 FBS free medium for 12 hours, and treated with IL-6 for 15 minutes. GCK, p-MKK4, p-MKK7, and p-JNK was detected by western blot and β-actin was detected as loading control. (F) Transduced and selected MM.1S (Tet-on-shGCK) cells were cultured with Dox (400 ng/mL) for 2 days, then treated with IL-6 and Dox for 3 days. Cell proliferation was detected by MTS assay.