TAPP2 KD impairs leukemic cell migration into bone marrow stromal cell layers.

<p>NALM-6 cells were added to a confluent layer of S17 stromal cells and allowed to migrate for 10 hours. Under-stroma migration was quantified as percentage of total input cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057809#s2" target="_blank">Methods</a>. <b>A.</b> Under-stroma migration is pertussis toxin sensitive and partially blocked by PI3K inhibtion. PT: addition of 0.1 µg/ml pertussis toxin (GPCR inhibitor); GDC: addition of 2 µM GDC-0941 (pan PI3K inhibitor); DMSO, vehicle control for GDC-0941. <b>B.</b> Under-stroma migration is partially dependent on the SDF-1 receptor CXCR4. Control, not treated; AMD, 10 or 100 µM AMD3100 (CXCR4 inhibitor); SDF, 0.1 or 1 µg/ml SDF-1 (to desensitize CXCR4). Results are mean ± SD of three independent experiments. Significant difference in leukemic migration into the stromal layer was determined by Student’s <i>t</i> test: **p < 0.01 or ***p<0.001. <b>C.</b> Phase contrast imaging of the under-stroma migration of control or TAPP2 KD NALM-6 cells into S17 stromal cells. Migrated cells, indicated by black arrows, are characterized by the dark round appearance in contrast to the stromal cells, indicated by white arrows. Images represent two independent experiments. <b>D.</b> TAPP2 KD inhibits under-stroma migration. NALM-6 cells were partially transduced with GFP-expressing pSIH1-H1-copGFP control lentivirus or TAPP2 KD lentivirus. Migration into S17 or M2-10B4 stromal layers was normalized as the migration of GFP+ (transduced) cells relative to that of GFP- cells (internal control) in the same sample. Results from one experiment performed in replicates were shown as mean ± SD, representing two independent experiments confirming migration inhibition by TAPP2 KD. Significant difference between control and KD cells based on each stromal type was calculated with Student <i>t</i> test: *p<0.05.