Deficiency of FLOT2 impairs the cytoskeleton in BCR-ABL1⁺, but not MLL-AF9⁺ cells. (A-B) Percentage of oncogene-positive (GFP⁺) lineage-negative (Lin⁻) apoptotic cells of all GFP⁺ Lin⁻ cells from the BM of WT mice transplanted with WT or Flot2 KO BCR-ABL1- (n.s.; t test; n = 7-8) (A) or MLL–AF9-positive BM (n.s.; t test; n = 7) (B), detected by annexin V staining. (C-D) Cell cycle analysis of oncogene-positive (GFP⁺) lineage (Lin) negative cells of all GFP⁺ Lin⁻ cells from the BM of WT mice transplanted with WT or Flot2 KO BCR–ABL1- (n.s.; ANOVA; Tukey test; n = 8) (C) or MLL–AF9-positive BM (n.s.; ANOVA; Tukey test; n = 13) (D). (E) Immunofluorescence staining showing the localization of myosin IIa in uropods of WT or Flot2 KO BCR-ABL1⁺ Lin- cells treated with vehicle or CXCL12 (1 ng/μL) for 4 hours. The images are representative of 3 independent experiments. The scale bar represents 25 µm. (F) Quantification of the percentage of myosin IIa staining in uropods in WT (red) or Flot2 KO (blue) Lin- cells per image as in (E) (P = .0001 [WT]; n.s. [Flot2 KO]; P = .0156 [WT vs Flot2 KO]; ANOVA; Tukey test; n = 7-12). (G) Immunofluorescence staining showing the localization of myosin IIa in uropods of WT or Flot2 KO MLL-AF9⁺ Lin- cells treated with vehicle or CXCL12 (1 ng/μL) for 4 hours. The images are representative of 3 independent experiments. The scale bar represents 25 µm. (H) Quantification of the percentage of myosin IIa staining in uropods in WT (red) or Flot2 KO (blue) Lin- cells per image as in (G) (P = .0611 [WT]; n.s. [Flot2 KO]; ANOVA; Tukey test; n = 8-12). (I) Immunofluorescence studies on 3T3 fibroblasts transduced with BCR–ABL1- or MLL–AF9-expressing retrovirus and either Scrambled- or Flot2 shRNA-expressing lentivirus, grown on coverslips and stained with phalloidin. The white arrows point to examples of the impaired cytoskeleton. Note that the cytoskeleton is known to be impaired in BCR-ABL1⁺ cells.³⁰  The images are representative of 3 independent experiments. The scale bar represents 25 µm. (J) Quantification of the immunofluorescence staining in (I). The average number of phalloidin⁺ aggregates representing an impaired cytoskeleton per cell was quantified (P < .0001 [BCR-ABL1]; ANOVA; Tukey test; n = 12-15).