Figure 4.

ST3Gal4 Knockout in Human Leukocytes Abolishes Selectin-Dependent Leukocyte Rolling

(A) HL-60s were stably transduced with SiC-V2-Cas9G7 virus to create Cerulean-positive cells. These were subsequently transduced with SiC-V1 virus carrying either scramble-sgRNA or ST3Gal4-sgRNA. Following sorting of dTomato⁺Cerulean⁺ cells at day 3, 1 μg/mL Dox was added from days 4 to 14. (B) The Surveyor assay monitored ST3Gal4 (top) and Cas9 (bottom) indels. (C) Fluorescence microscopy at day 14 measured dTomato reporter. Dox treatment resulted in Cas9 editing and loss of red fluorescence. (D) Flow cytometry measured cell surface sialyl Lewis-X expression (using mAb HECA-452), and also P-, E-, and L-selectin binding function (treatments same as C). Loss of ST3Gal4 activity reduced sLe^X expression and selectin binding (events in bottom-left quadrant of each sub-plot). (E) No Dox cells from (C) were perfused over selectin substrates in microfluidic flow cell at wall shear stress of 2 dynes/cm². Cells lacking ST3Gal4 displayed minimal interaction with selectin substrates in bright field. Controls used function blocking mAbs against P- (clone G1), E- (P2H3), and L- (DREG-56) selectin. See also Figure S4.