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. 2003 Aug;23(16):5556–5571. doi: 10.1128/MCB.23.16.5556-5571.2003

FIG. 5.

FIG. 5.

Loss of Snk/Plk2 sensitizes tumor cells to spindle inhibitors through apoptosis.

(a) (Left panel) U2OS cells and HeLa cells were transfected with Snk/Plk2 V5-six-histidine tag (4.5 μg), pEGFP (0.5 μg), and control siRNA duplex or Snk/Plk2 siRNA duplex (200 nmol). After 48 h, cells were harvested for protein and analyzed for expression with anti-V5 antibody. Green fluorescent protein was used to control for possible differences in transfection efficiencies. Actin was used as to confirm that an equivalent amount of protein was loaded in each lane. (Right panel) U2OS cells were transfected with control siRNA duplex or Snk/Plk2 siRNA duplex (300 nmol). After 48 h, cells were harvested for total RNA,and Snk/Plk2 mRNA levels were determined by a TaqMan real-time quantitative RT-PCR assay. All expression levels are normalized to GAPDH in each well, and changes are defined as levels of Snk/Plk2 mRNA with Snk/Plk2 siRNA duplex divided by levels of Snk/Plk2 mRNA with control siRNA duplex. (b) U2OS cells were transfected with control siRNA duplex or Snk/Plk2 siRNA duplex (300 nmol). Twenty-four hours after transfection, cells were treated with 1 μM paclitaxel. After 24 or 48 h of paclitaxel treatment, cells were harvested and collected for fluorescence-activated cell sorting analysis. Cell death was scored as the percentage of cells with a DNA content of less than 2N. (c) Quantitation of experiment in panel b. Depicted in the chart is one of several experiments each done in triplicate. (d) The osteosarcoma cell line U2OS, the non-small cell lung cancer cell line H460, and the cervical carcinoma cell line HeLa, were transfected with control siRNA duplex or Snk/Plk2 siRNA duplex (300 nmol). Twenty-four hours after transfection, cells were treated with nocodazole (1 μg/ml). After 24 h of treatment, cells were harvested and collected for fluorescence-activated cell sorting analysis. Cell death was scored as the percentage of cells with a DNA content of less than 2N. Depicted in the chart is one of several experiments, each done in triplicate.