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. 2013 Feb 27;8(2):e57809. doi: 10.1371/journal.pone.0057809

Figure 1. TAPP2 knockdown impairs SDF-1-induced migration. A.

Figure 1

NALM-6 cells migration towards SDF-1 is impaired by PI3K inhibitors. NALM-6 cells were treated with drug vehicle (DMSO) or PI3K inhibitors GDC-0941, CAL-101, AS-605240 or TGX-221 (all 2 µM), and migration was assayed in transwell chambers containing 100 ng/ml SDF-1. Graphs represent the mean ± SD from three independent experiments. All PI3K inhibitors significantly reduced the migration compared to vehicle control according to Student’s t test: *p<0.05, **p<0.01 and ***p<0.001. B. TAPP2 knockdown (KD) inhibits NALM-6 migration on a variety of substrata. NALM-6 cells were transduced with control pSIH1-H1-copGFP lentivirus (Con) or virus expressing human TAPP2-targeting shRNA (KD). Western blot represents one of four batches of transduction confirming TAPP2 KD. Transwell assays of migration to 100 ng/ml SDF-1 were performed, where the insert was either uncoated polycarbonate (PC) or coated with 10 µg/ml laminin or fibronectin. Results of three independent experiments are shown as mean ± SD. Significance comparing control versus KD cell migration was calculated for each coating condition using Student’s t test: *p<0.05. Note the effect of TAPP2 KD on migration in uncoated transwell chambers has been reproduced in 5 additional experiments. C. TAPP2 KD does not significantly affect cell viability. Control or TAPP2 KD NALM-6 cells were stained for Annexin V and with DAPI and analyzed by flow cytometry. Percentages (mean ± SD) of viable cells (Annexin V and DAPI double negative) from four independent experiments are shown. Student t test found no significant difference in cell viability. D. TAPP2 KD does not significantly affect surface expression of CXCR4. Cells were stained for surface CXCR4 and analyzed by flow cytometry. CXCR4 levels were expressed as percentage of mean fluorescence intensity (MFI) relative to control cells. Bars represent mean ± SD of two independent experiments. E. TAPP2 KD inhibits SDF-1-induced migration in combination with PI3K inhibitors. Control or TAPP2 KD NALM-6 cells were assayed for migration to SDF-1 in the presence of vehicle (DMSO) or PI3K inhibitors (2 µM) GDC-0941, CAL-101 or TGX-221. Results are mean ± SD of three independent experiments. Significance of difference in migration was confirmed by Student’s t test comparing PI3K inhibitor alone versus the corresponding TAPP2 KD+PI3K inhibitor group: ***p<0.001. Note all TAPP2 KD+PI3K inhibitor groups were also significantly different than TAPP2 KD alone (all p<0.01 by Student’s t test).