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P.S. Godavarthy et al.
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Figure 4. Binding of CD44 to E-selectin influences the cell cycle of BCR-ABL1+ cells. (A) Representative two-photon in vivo microscopy image of the calvarial bone marrow of an unirradiated Tie2-GFP mouse injected with 1×106 sorted BCR-ABL1+ BaF3 cells, labeled with CMTMR (orange), 2 h prior to in vivo microscopy. Bones were visualized in blue due to second harmonic generation. The scale bar represents 50 mm. (B) Time of contact (seconds), determined by in vivo microscopy as in (A) between the calvarial endothelium and BCR-ABL1+ or BCR-ABL1+ CD44 overexpressing BaF3 cells labeled with CMTMR and injected into unirradiated Tie2-GFP mice (P<0.001, t-test, n=3). (C) Cell cycle analysis by Ki67-staining of serum-starved BaF3 cells coexpressing BCR-ABL1 and CD44 plated on E-selectin-coated plates and treated with vehicle, GMI-1271, imatinib or the combination of GMI-1271 plus imatinib. The percentages of GFP+ (BCR-ABL1+) cells of total in the G0, G1 or G2-S-M phases of the cell cycle are shown. The differences are not statistically significant. (D, E) Cell cycle analysis by Ki67-staining of serum-starved non-adherent (D) or adherent (E) BCR-ABL1+ BaF3 cells plated on E- selectin-coated plates and treated with vehicle, GMI-1271, imatinib or the combination of GMI-1271 plus imatinib. The percentages of GFP+ (BCR-ABL1+) cells of total in the different phases of the cell cycle are shown. Seventy thousand cells were plated and allowed to adhere for 6 h (n=3). (F, G) Corrected total cell fluorescence (CTCF) for CDK6 (F) and p16 (G) in BCR-ABL1+ BaF3 cells plated on E-selectin in the adhesion assay, performed in the presence of vehicle or GMI-1271. Seventy thousand cells were plated (n=3). (H) Cell cycle analysis by Ki67-staining of GFP+ (BCR-ABL1+) Lin- c-Kit+ cells from Balb/c mice with established chronic myeloid leukemia treated with vehicle, GMI-1271, imatinib or the combination of GMI-1271 plus imatinib on day 14 after transplantation. The percentages of GFP+ (BCR-ABL1+) Lin- c-Kit+ cells of total in the G0, G1 or G2-S-M phases of the cell cycle are shown (P values as indicated, analysis of variance; Tukey test, n=5).
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haematologica | 2020; 105(1)