PAK4 triggers MEK/ERK pathway activation in MM cells. (A) WB analysis of PAK4 expression in RPMI 8226 cells overexpressing PAK4. (B) Effect of PAK4 overexpression in RPMI 8226 cells was evaluated over time by (3H)-thymidine uptake and presented as fold change increase compared with day 1. (C) In vivo evaluation of the effects of PAK4 overexpression on MM cells. Growth curve assesses tumor size after injection of an equal number of PAK4 overexpression or empty vector cells subcutaneously into the right posterior flank region of severe combined immunodeficiency mice. Data are shown as the mean values ± standard deviation. (D) PAK4 mRNA levels were evaluated in control and PAK4-overexpressing cells by qPCR analysis. Data are presented as fold change increase from corresponding control cells. (E) Effect of PAK4 overexpression in H929, MM1S, INA6, and KMS12BM MM cell lines was evaluated over time by (3H)-thymidine uptake and presented as fold change increase compared with day 1. (F) WBs showing inhibition of indicated signaling proteins in H929 and RPMI 8226 myeloma cells ectopically expressing PAK4, compared with respective control. GAPDH was used as loading control. One representative blot of 2 is shown. (G) Control and ectopically expressing PAK4 cells were treated with and without U0126 (10 µM) or LY29004 (10 µM) for 48 hours. Cell proliferation was assessed by (3H)-thymidine uptake and presented as count per minute (CPM). (H) MM1S cells transfected with inducible tetracycline-inducible pTRIPz-Turbo-RFP vector #23 or control were treated with 2.5 μg/mL doxycycline for 3 consecutive days. WB analysis was performed using indicated mAbs. *P < .05; **P < .005; ***P < .0005. CNT, control; NS, not significant; WT, wild type.
