HCELL expression is specifically reduced on KG1a cells by CD44 siRNA transduction. (A) Flow cytometric analysis of CD44, PSGL-1, and integrin expression of untreated, vector-transduced (control), and CD44 siRNA-transduced KG1a cells. CD44 expression was reduced on CD44 siRNA transduction, as measured by anti-CD44 mAb 2C5 (used in Figure) and further confirmed using anti-CD44 mAb 515 (data not shown, which recognizes an epitope distinct from 2C5). PSGL-1, VLA-2 (CD49b/CD29) integrin, and VLA-4 (CD49d/CD29) integrin expression was unaffected by viral transduction. Values are mean fluorescence intensities ± SEM; n = 6. *P < .05 relative to untreated and vector controls. (B-C) Western blot analysis of HCELL on KG1a cells. Whole cell lysates equivalent to 2 × 105 (for anti-CD44 mAb, 2C5) and 1 × 105 (for HECA-452) untreated, vector-transduced, and CD44 siRNA-transduced KG1a cells were resolved on a 7.5% SDS-PAGE gel under reducing conditions and immunoblotted with the appropriate mAb. Note that HCELL expression is nearly eliminated by CD44 silencing (minimal staining of HECA-452 and CD44 mAb at ∼ 100 kDa in CD44-silenced cell lysates relative to untreated and vector-transduced cell lysates).
