Silencing CD34 leads to a decrease in microvilli and higher rolling velocities with increasing shear stress. (A) Flow cytometric analysis of E-selL expression and E-Ig and HECA-452 binding of scrambled and CD34 siRNA-nucleofected KG1a cells (CD34-KD). This is a representative figure of n = 4 independent experiments depicting the geometric mean fluorescent intensity (G.MFI). (B) Equivalent amounts of scrambled and CD34-KD cell lysates were subjected to western blot analysis and stained for CD34, CD44, CD43, PSGL-1, sLe^x (HECA-452), and E-Ig. Blots are representative of n = 4 independent experiments. (C) Scrambled or CD34-KD KG1a cells were each perfused over CHO-E cell monolayers for 1 minute at 0.28 dyne/cm², and then detachment assays were employed by increasing the shear stress stepwise every 15 seconds. The average number of rolling cells in 4 distinct fields of view for each experiment (n = 4) was counted. (D) Single-cell tracking with Imaris V7.6.4 software was used to calculate KG1a rolling velocity over CHO-E cells from (C) at each shear stress depicted as described. The adhesion histogram is representative of n = 4 independent experiments, and data are reported as the mean ± SEM (error bars). *P < .05. (E) KG1a cells were either transfected with scrambled control siRNA (scrambled control) or with CD34 siRNA (CD34-KD), and 48 hours later, cells were fixed with glutaraldehyde and prepared for TEM analysis as outlined in supplemental Materials and methods. Red arrows point out some of the microvilli-type structures that are evident in the scrambled control but not in the CD34-KD images. These are representative cells of over 30 different cells imaged under each condition.