Knockdown of FLOT2 in human leukemia cell lines impacts leukemia cell physiology and function. (A-B) Relative adhesion of K562 (P = .0006; t test; n = 4) (A) or THP1 cells (P = .0029; t test; n = 4) (B), transduced with Scrambled shRNA (red)- or FLOT2 shRNA (blue)-expressing lentivirus to E–selectin-coated plates; 7 × 10⁴ cells had been plated and allowed to adhere for 6 hours. (C-D) Transwell migration of K562 (P = .0002; t test; n = 3) (C) or THP1 (P = .0112.; t test; n = 3) (D) cells transduced with Scrambled shRNA (red)- or FLOT2 shRNA (blue)-expressing lentivirus toward HS-5 stromal cells. Leukemia cells (1.5 × 10⁵) had been plated and allowed to migrate for 16 hours through an 8 µm pore size transwell. (E) Coimmunoprecipitation of lysates of K562 (left) vs THP1 (right) cells with anti-CD44 and anti-FLOT2 antibodies. The subsequent Western blot was performed with an antibody to CD44 (82 kDa) and FLOT2 (49 kDa). The blots are representative of 3 independent replicates. (F) Immunoblot showing the expression of CD44 (82 kDa) and GAPDH (37 kDa) as loading control in lysates of K562 and THP1 cells transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus. The blot is representative of 5 independent experiments. (G-H) Relative mean fluorescence intensity of CD44 on K562 (P = .0001; t test; n = 5) (G) and THP1 (P = .0145; t test; n = 5) (H) cells, transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus. (I) Immunoblot of cell lysates from a cell surface biotinylation assay on K562 or THP1 cells, transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus. Total protein samples (input) and samples containing biotinylated cell surface proteins and immunoprecipitated with streptavidin-binding beads (IP-Biotin) were probed with an antibody to CD44 (82 kDa) and GAPDH (37 kDa). The images are representative of 2 independent experiments. (J-K) Membrane fractions of K562 (J) and THP1 (K) cells transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus. The lipid raft protein caveolin (46 kDa) and CD44 (82 kDa) were detected by immunoblotting. The numbers represent the different membrane fractions obtained via a sucrose gradient. (L-M) Relative adhesion of K562 (P = .008; t test; n = 3) (L) or THP1 cells (n.s.; t test; n = 3) (M), transduced with Scrambled shRNA (red)- or FLOT2 shRNA (blue)-expressing lentivirus, to HS-5 stromal cells; 7 × 10⁴ cells had been plated and allowed to adhere for 6 hours.