PAK4 expression affects growth and survival in MM. (A) Protein lysates from a panel of MM cell lines were analyzed for PAK4 and p-PAK4 expression by WB. GAPDH was used as loading control. One representative experiment of 2 is shown. (B) PAK4 and p-PAK4 relative expression levels. Data reported represent mean of 3 independent experiments. (C) Representative images of PAK4 and p-PAK4 immunocytochemistry stain in BM from normal, SMM, and symptomatic MM individuals. Scale bars: 100 μm and 5 μm. (D) Genetic depletion of PAK4 was achieved using 4 different tetracycline-inducible pTRIPz-Turbo-RFP vectors (Thermo Scientific, Pittsburgh, PA) containing the target sequence or scrambled control. Transfected MM1S cells were plated in growth medium in the absence or presence of 2.5 μg/mL doxycycline. qPCR analysis (right panel) was performed at day 3, confirming decreased PAK4 mRNA levels in cells expressing inducible PAK4 shRNAs compared with scrambled cells. (E) Cellular proliferation was evaluated by (3H)-thymidine uptake and presented as growth rate increase compared with t = 0. In medium containing doxycycline, reduced expression of PAK4 is accompanied by a reduction of cell growth rate compared with control cells. (F) Apoptosis was evaluated after 3 days of induction with 2.5 μg/mL doxycycline, using Annexin V and propidium iodide (PI) staining followed by flow cytometry acquisition and analysis. (G) Caspases activation was evaluated after 3 days of induction with 2.5 μg/mL doxycycline by luminescence assay.
