Ectopic expression of catalytic domain HMGCR-FL, but not HMGCR-D13, reduces sensitivity to lovastatin in sensitive MM cells. (A) Schematic of the ectopic HMGCR catalytic domain constructs. (B) KMS11 and LP1 cell lines ectopically expressing the empty GFP vector control, cHMGCR-FL, or cHMGCR-D13 constructs were generated and assessed for transcript expression of HMGCR-FL (left), HMGCR-D13 (middle), and total endogenous HMGCR (right) using primers i, ii, and iii (Figure 2A), respectively, by real-time PCR, measured relative to GAPDH. (C) KMS11 cells ectopically expressing the empty GFP vector control, cHMGCR-FL, cHMGCR-D13, or BCL2 were assessed for protein expression with anti-HMGCR, anti-BCL2, and antiactin as a loading control. (D) KMS11 cells expressing the cHMGCR constructs were exposed to increasing concentrations of lovastatin in an MTT assay to measure cell viability (left). Only the cells expressing cHMGCR-FL demonstrated an increase in their MTT50 for lovastatin, the concentration that is required to reduce viability of the population by 50% (right). (E) Cells expressing the vector control, cHMGCR-FL, cHMGCR-D13, or BCL2 were also exposed to increasing concentrations of lovastatin (left), melphalan (middle), or bortezomib (right) and assayed for the proportion of their pre-G1 populations by fixed PI. *P < .05 (Student t test with Welch adjustment for multiple testing comparing expression in the ectopic construct expressing cells with the GFP control). All experiments were performed a minimum of 3 times. Data are mean ± SD.
