Figure 7.

Pharmacological blockage of GCK inhibits MM cell proliferation and induces cell death. (A) RAS^WT MM cell lines (KMS12-PE, OPM2, LP-1, SKMM-2, and U266) and RAS^Mut MM cell lines (MOLP-8, RPMI-8226, H929, MM.1S, and JJN3) were incubated with TL4-12 for 4 days. Cell proliferation was analyzed by MTS assay. Data represent the mean ± SD of n = 3 experiments. (B) RAS^WT LP-1 and K-RAS^G12A MM.1S cells were incubated with TL4-12 for 24 hours to analyze IKZF1, c-MYC, BCL-6, and P53 expression by western blotting. β-actin was detected as loading control. (C) Cells were treated by TL4-12 for 4 days to detect cell apoptosis with Annexin V and 7-AAD staining by flow cytometry assay. (D) MM.1S cells were treated with TL4-12 at the indicated concentrations with or without PS341 (1μM) for 24h. Lysates were analyzed by western blotting to compare the levels of IKZF1, c-MYC, and IRF-4. β-actin expression was probed for loading control. (E-F) N-RAS^G13D H929 cells were transduced using a lentivirus with control shRNA (shCNTL) or CRBN-shRNA (shCRBN). GFP-sorted cells were cultured in the presence of LEN or TL4-12 for 24 hours to analyze IKZF1, CRBN expression by western blotting (E); and for 4 days for cell proliferation detected by MTS assay (F). (G) Transduced and selected Tet-On-shGCK-MM.1S and Tet-On-shGCK-RPMI-8266 cells were cultured with LEN at the indicated concentrations with or without Dox 400 ng/mL for 3 days and cell proliferation was measured by MTS assay,