Figure 7.
CDC42 is differentially expressed, regulated, and localized in FLOT2-deficient CML cells. (A) Immunoblot of lysates from K562 (left) vs THP1 (right) cells, transduced with either Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus, probed with antibodies to CDC42 (42 kDa) or GAPDH (37 kDa). The images are representative of 4 independent experiments. (B-C) Immunofluorescence staining for CDC42 in K562 (B) or THP1 (C) cells, transduced with either Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus. The images are representative of 3 independent experiments. The scale bar represents 10 µm. (D) Coimmunoprecipitation of lysates of K562 (left) and THP1 (right) with PAK1-protein binding beads (PAK1 PDB) as part of a CDC42 activation assay. PAK1 was used as a downstream effector of CDC42 to isolate the active GTP-bound form of CDC42. The Western blot was performed using an anti-CDC42 antibody (42 kDa). (E-F) Immunoblot of lysates from K562 (left) vs THP1 (right) cells, transduced with either Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus, probed with antibodies to PAK1 (68 kDa) (E), pPAK1 (70 kDa) (F), and GAPDH (37 kDa). The images are representative of 5 independent experiments. (G-H) Relative adhesion of K562 (ANOVA; Tukey test; n = 4) (G) or THP1 (ANOVA; Tukey test; n = 6) (H) cells, transduced with sh Scrambled- or FLOT2 shRNA-expressing lentivirus and treated with vehicle or the CDC42 inhibitor ML141 (5 nM) to HS-5 stromal cells. The cells were allowed to adhere for 6 hours. (I-J) Immunofluorescence staining (I) and quantification (J) of F-actin using phalloidin in K562 cells, transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus and treated with vehicle or the CDC42 inhibitor ML141 (100 nM for 6 h). (K-L) Immunofluorescence staining (K) and quantification (L) of F-actin using phalloidin in THP1 cells, transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus and treated with vehicle or the CDC42 inhibitor ML141 (100 nM for 6 h). (M-N) Percentage of total leukocytes (P = .0578; t test; n = 5) (M) and absolute number per femur of GFP+ (BCR-ABL1+) WT LKS cells (P = .0221; t test; n = 5) (N), which had been pretreated ex vivo with vehicle (red) or the CDC42 inhibitor ML141 (5 µM for 4 hours) (blue) and homed to the BM of WT mice 18 hours after transplantation. (O) Percentage of BCR-ABL1+ (GFP+) CD11b+ myeloid cells of total leukocytes in the PB (day 13 after transplantation) of mice transplanted with WT BM transduced with BCR–ABL1-expressing retrovirus and ex vivo pretreated with vehicle (red) or the CDC42 inhibitor ML141 (blue) (5 µM for 4 hours) before transplantation, (P = .0405; t test; n = 5). (P) Kaplan–Meier-style survival curve of WT recipient mice transplanted with WT BM transduced with BCR–ABL1-expressing retrovirus and ex vivo pretreated with vehicle (solid line, red) or the CDC42 inhibitor ML141 (dotted line, blue) (5 µM for 4 hours) before transplantation (P = .0147; log-rank test; n = 6).

CDC42 is differentially expressed, regulated, and localized in FLOT2-deficient CML cells. (A) Immunoblot of lysates from K562 (left) vs THP1 (right) cells, transduced with either Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus, probed with antibodies to CDC42 (42 kDa) or GAPDH (37 kDa). The images are representative of 4 independent experiments. (B-C) Immunofluorescence staining for CDC42 in K562 (B) or THP1 (C) cells, transduced with either Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus. The images are representative of 3 independent experiments. The scale bar represents 10 µm. (D) Coimmunoprecipitation of lysates of K562 (left) and THP1 (right) with PAK1-protein binding beads (PAK1 PDB) as part of a CDC42 activation assay. PAK1 was used as a downstream effector of CDC42 to isolate the active GTP-bound form of CDC42. The Western blot was performed using an anti-CDC42 antibody (42 kDa). (E-F) Immunoblot of lysates from K562 (left) vs THP1 (right) cells, transduced with either Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus, probed with antibodies to PAK1 (68 kDa) (E), pPAK1 (70 kDa) (F), and GAPDH (37 kDa). The images are representative of 5 independent experiments. (G-H) Relative adhesion of K562 (ANOVA; Tukey test; n = 4) (G) or THP1 (ANOVA; Tukey test; n = 6) (H) cells, transduced with sh Scrambled- or FLOT2 shRNA-expressing lentivirus and treated with vehicle or the CDC42 inhibitor ML141 (5 nM) to HS-5 stromal cells. The cells were allowed to adhere for 6 hours. (I-J) Immunofluorescence staining (I) and quantification (J) of F-actin using phalloidin in K562 cells, transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus and treated with vehicle or the CDC42 inhibitor ML141 (100 nM for 6 h). (K-L) Immunofluorescence staining (K) and quantification (L) of F-actin using phalloidin in THP1 cells, transduced with Scrambled shRNA- or FLOT2 shRNA-expressing lentivirus and treated with vehicle or the CDC42 inhibitor ML141 (100 nM for 6 h). (M-N) Percentage of total leukocytes (P = .0578; t test; n = 5) (M) and absolute number per femur of GFP+ (BCR-ABL1+) WT LKS cells (P = .0221; t test; n = 5) (N), which had been pretreated ex vivo with vehicle (red) or the CDC42 inhibitor ML141 (5 µM for 4 hours) (blue) and homed to the BM of WT mice 18 hours after transplantation. (O) Percentage of BCR-ABL1+ (GFP+) CD11b+ myeloid cells of total leukocytes in the PB (day 13 after transplantation) of mice transplanted with WT BM transduced with BCR–ABL1-expressing retrovirus and ex vivo pretreated with vehicle (red) or the CDC42 inhibitor ML141 (blue) (5 µM for 4 hours) before transplantation, (P = .0405; t test; n = 5). (P) Kaplan–Meier-style survival curve of WT recipient mice transplanted with WT BM transduced with BCR–ABL1-expressing retrovirus and ex vivo pretreated with vehicle (solid line, red) or the CDC42 inhibitor ML141 (dotted line, blue) (5 µM for 4 hours) before transplantation (P = .0147; log-rank test; n = 6).

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