Figure 3.

ARK5 depletion inhibits multiple myeloma cell growth via Rb/mTOR/MYC pathways and induces apoptosis and cell-cycle arrest. A, ARK5 repression significantly attenuated phosphoS6K, phosphoRb, MYC, and acetyl MYC activities in multiple myeloma. Conversely, depletion of ARK5 increased AMPK and SIRT1. Western blot analysis showing the levels of ARK5, SIRT1, MYC, acetyl MYC, total S6K, Rb, phosphoS6K, and phosphoRb proteins after 24-hour transfection in MM1.S and NCI-H929 siRNA–transfected cells. There was no difference in total Rb and total S6K between the groups. B and C, MM1.S and NCI-H929 cells were subjected to SIRT1 depletion by siRNA and SIRT1 activation with SRT1720. Western blotting confirmed that depletion of SIRT1 increased the acetylation activities of MYC, whereas the activation with SRT1720 resulted in decreased acetylation of MYC in a dose- and time-dependent manner. D, transfection in MM1.S and NCI-H929 cells induced apoptosis in ARK5-depleted cells as compared with nontransfected cells after 24-hour transfection confirmed with Annexin V/PI double staining. E, transfection in MM1.S and NCI-H929 cells also induced cell-cycle arrest in ARK5-depleted cells confirmed by EdU cell proliferation assay. Forty percent of MM1.S cells transfected with scrambled control siRNA showed EdU incorporation. EdU incorporation in cells transfected with ARK5 siRNA was approximately half that of the negative control. In NCI-H929 cells, 50% of cells transfected with negative control siRNA showed EdU incorporation, and EdU incorporation in cells transfected with ARK5 siRNA was less than half that.