Figure 4. TRIP13 knockdown inhibits myeloma cell growth, induces apoptosis and lowers tumor burden in vivo.

A. TRIP13 knockdown was identified in myeloma cell lines ARP1 and OCI-My5 (TRIP13-ShRNA) compared to control cells that were non-target scramble transfected (Scramble). B. TRIP13-ShRNAMM cell lines ARP1, OCI-My5 as well as their scrambles were counted by trypan blue exclusion for consecutive 5 days for cell growth. Results from 3 independent experiments are shown (*p < 0.05). C. PARP and cleaved caspase 3 (C3) were determined using western blot in TRIP13-ShRNAmyeloma cell lines ARP1 and OCI-My5 and their scrambled controls (Scr) 48 h post Doxycycline (Dox) treatment. D. Representative images of anchorage-independent colony formations of TRIP13-ShRNA myeloma cell lines ARP1 and OCI-My5 and their scrambled controls (Scramble) (magnification×4). E. Representative images of overall colony formation for TRIP13-ShRNA myeloma cell lines ARP1 and the scrambled control (Scramble) in the each well (left panel). Overall colony numbers of each well were quantified and the percentage of TRIP13-ShRNA/Scramble was calculated. Results from 3 independent experiments are shown (*p<0.05) (right panel). F. Dox treatment induced effective TRIP13 knockdown in tumor masses of mice injected with TRIP13-ShRNA myeloma cell line OCI-My5 compared to those with scrambled control (Scramble). G. 1.5×10⁶ TRIP13-ShRNA OCI-My5 cells or the scrambled control (Scramble) were injected subcutaneously into the abdomen of NOD-Rag/null gamma mice. TRIP13 knockdown was induced by the addition of Dox to the drinking water 16 days after injection. Tumor growth was assessed for each group every four days (*p < 0.05). H. Mice were properly sacrificed and differences in tumor size were shown between the two groups of mice on Day 32 post injection. I. Tumor volume was quantified and compared between the two groups of mice (*p<0.05).