A largely disrupted CSF1R JMD in GC dictated the megakaryocytic phenotype. (A) CSF1R fusions identified in B-ALL and AMKL. The full length of CSF1R contains 972 amino acids (AAs). JMD is located between AAs 542 and 574. CSF1R fusions identified in B-ALL and AMKL are shown in the box. Ig, immunoglobulin domains. (B) Schematic representation of the lentiviral vectors. The arrows indicate different CSF1R breakpoints. Trunc, truncated. (C) Flow cytometric analysis of CD markers in M07E and TF1 cells transduced with the indicated lentivirus for 72 hours. Transduced (EGFP+) cells were gated for analysis. Left, representative results of 3 independent experiments. Right, MFI quantification. ∗ and ∗∗∗∗ indicate P < .05 and P < .0001 vs EV, respectively, by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (D) Flow cytometric analysis of ERK1/2 and STAT5 phosphorylation in M07E cells after GC and GC543 overexpression. M07E cells were transduced with EV, GC, and GC543 lentiviruses for 72 hours (0h). Subsequently, the cells were washed in phosphate-buffered saline 3 times and cultured in medium without cytokine (GM-CSF) for 4 hours (4h) and 8 hours (8h). Transduced (EGFP+) cells were gated for analysis. Left, representative results of 3 independent experiments. Right, MFI quantification. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ indicate P < .05, P < .01, P < .001, and P < .0001 vs EV, respectively, by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (E) Heat map of GC-upregulated genes overlapped in coagulation hallmark gene set (systematic name M5946, which consists of genes encoding components of blood coagulation system and is also upregulated in platelets). Average expression values from the biological duplicates are shown. (F) Heat map of GC-downregulated genes overlapped in heme metabolism hallmark gene set (systematic name M5945, which includes genes related to the metabolism of heme, a cofactor consisting of iron and porphyrin, as well as erythroblast differentiation). Average expression values from the biological duplicates are shown. (G) RT-qPCR analysis of KLF1 mRNA levels in M07E and TF1 cells transduced with EV, GC, and GC543 lentiviruses. ∗ and ∗∗∗∗ indicate P < .05 and P < .0001 vs EV, respectively, by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (H) Western blotting analysis of KLF1 protein levels in M07E and TF1 cells transduced with EV, GC, and GC543 lentiviruses. Representative images of 3 independent experiments. (I) Left, schematic representation of the luciferase reporter vectors. Right, K562 cells were cotransfected with the reporter and overexpression vectors (LeGOiG2-EV, LeGOiG2-GC, or LeGOiG2-GC543). The reporter activity was measured by Nano-Glo Dual-Luciferase Reporter Assay 48 hours after transfection. ∗ indicates P < .05 vs EV by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (J) Left, schematic representation of the lentiviral vectors. Right, flow cytometric analysis of CD41a and CD61 in M07E cells cotransduced with the indicated lentivirus. Transduced (EGFP+ and mCherry+) cells were gated for analysis. ∗ and ∗∗ indicate P < .05 and P < .01 vs LeGOiC2-EV, respectively, by Student t test. Results are expressed as mean ± SD from 3 independent experiments.

A largely disrupted CSF1R JMD in GC dictated the megakaryocytic phenotype. (A) CSF1R fusions identified in B-ALL and AMKL. The full length of CSF1R contains 972 amino acids (AAs). JMD is located between AAs 542 and 574. CSF1R fusions identified in B-ALL and AMKL are shown in the box. Ig, immunoglobulin domains. (B) Schematic representation of the lentiviral vectors. The arrows indicate different CSF1R breakpoints. Trunc, truncated. (C) Flow cytometric analysis of CD markers in M07E and TF1 cells transduced with the indicated lentivirus for 72 hours. Transduced (EGFP+) cells were gated for analysis. Left, representative results of 3 independent experiments. Right, MFI quantification. ∗ and ∗∗∗∗ indicate P < .05 and P < .0001 vs EV, respectively, by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (D) Flow cytometric analysis of ERK1/2 and STAT5 phosphorylation in M07E cells after GC and GC543 overexpression. M07E cells were transduced with EV, GC, and GC543 lentiviruses for 72 hours (0h). Subsequently, the cells were washed in phosphate-buffered saline 3 times and cultured in medium without cytokine (GM-CSF) for 4 hours (4h) and 8 hours (8h). Transduced (EGFP+) cells were gated for analysis. Left, representative results of 3 independent experiments. Right, MFI quantification. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ indicate P < .05, P < .01, P < .001, and P < .0001 vs EV, respectively, by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (E) Heat map of GC-upregulated genes overlapped in coagulation hallmark gene set (systematic name M5946, which consists of genes encoding components of blood coagulation system and is also upregulated in platelets). Average expression values from the biological duplicates are shown. (F) Heat map of GC-downregulated genes overlapped in heme metabolism hallmark gene set (systematic name M5945, which includes genes related to the metabolism of heme, a cofactor consisting of iron and porphyrin, as well as erythroblast differentiation). Average expression values from the biological duplicates are shown. (G) RT-qPCR analysis of KLF1 mRNA levels in M07E and TF1 cells transduced with EV, GC, and GC543 lentiviruses. ∗ and ∗∗∗∗ indicate P < .05 and P < .0001 vs EV, respectively, by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (H) Western blotting analysis of KLF1 protein levels in M07E and TF1 cells transduced with EV, GC, and GC543 lentiviruses. Representative images of 3 independent experiments. (I) Left, schematic representation of the luciferase reporter vectors. Right, K562 cells were cotransfected with the reporter and overexpression vectors (LeGOiG2-EV, LeGOiG2-GC, or LeGOiG2-GC543). The reporter activity was measured by Nano-Glo Dual-Luciferase Reporter Assay 48 hours after transfection. ∗ indicates P < .05 vs EV by 1-way ANOVA followed by Dunnett test. Results are expressed as mean ± SD from 3 independent experiments. (J) Left, schematic representation of the lentiviral vectors. Right, flow cytometric analysis of CD41a and CD61 in M07E cells cotransduced with the indicated lentivirus. Transduced (EGFP+ and mCherry+) cells were gated for analysis. ∗ and ∗∗ indicate P < .05 and P < .01 vs LeGOiC2-EV, respectively, by Student t test. Results are expressed as mean ± SD from 3 independent experiments.

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