Figure 3.
FIP1L1 knockdown promotes t(8;21) leukemic cell differentiation. (A) Western blot showing FIP1L1 and actin (loading control) protein in Kasumi-1 cells after transduction with control shRNAs or shRNAs targeting FIP1L1. (B) GSEA plot comparing differentially expressed genes upon FIP1L1 knockdown in Kasumi-1 cells with the Brown Myeloid Cell Development UP gene set. The heatmap displays 15 genes in the leading edge of the gene set that are most upregulated upon FIP1L1 knockdown. (C) GSEA plot comparing differentially expressed genes upon FIP1L1 knockdown in Kasumi-1 cells with the Jaatinen Hematopoietic Stem Cell UP gene set. The heatmap displays 15 genes in the leading edge of the gene set that are most downregulated upon FIP1L1 knockdown. (D) CD34 cell surface expression measured by flow cytometry of Kasumi-1 cells 6 days after transduction with shRNAs targeting FIP1L1 or a control shRNA. Data are mean ± standard deviation (SD) of 3 independent experiments. ***P < .001, 1-way analysis of variance (ANOVA) with post hoc Tukey test. (E) Proliferation of Kasumi-1 cells after transduction with shRNAs targeting FIP1L1 or a control shRNA. Cells were seeded (day 0) after 2 days of puromycin selection. The graph displays the mean and SD of 3 technical replicates in 1 representative experiment of 2 independent experiments. **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. (F) Percentage of Annexin V–positive apoptotic Kasumi-1 cells 5 days after transduction with shRNAs targeting FIP1L1 or control shRNAs. Data are mean ± SD of 3 independent experiments. **P < .01, 1-way ANOVA with post hoc Tukey test. (G) Western blot showing FIP1L1 and actin (loading control) protein in Kasumi-1 cells after transduction with control sgRNAs targeting green fluorescent protein or sgRNAs targeting FIP1L1. (H) CD34 cell surface expression measured by flow cytometry of Kasumi-1 cells 10 days after transduction with sgRNAs targeting FIP1L1 or control sgRNAs. Data are mean ± SD of 3 independent experiments. **P < .01, Student t test. (I) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of various myeloid differentiation genes in Kasumi-1 cells transduced with sgRNAs targeting FIP1L1 or control sgRNAs. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. (J) RT-qPCR analysis of various HSC signature genes in Kasumi-1 cells transduced with sgRNAs targeting FIP1L1 or control sgRNAs. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. FDR, false discovery rate; MFI, mean fluorescent intensity; NES, normalized enrichment score.

FIP1L1 knockdown promotes t(8;21) leukemic cell differentiation. (A) Western blot showing FIP1L1 and actin (loading control) protein in Kasumi-1 cells after transduction with control shRNAs or shRNAs targeting FIP1L1. (B) GSEA plot comparing differentially expressed genes upon FIP1L1 knockdown in Kasumi-1 cells with the Brown Myeloid Cell Development UP gene set. The heatmap displays 15 genes in the leading edge of the gene set that are most upregulated upon FIP1L1 knockdown. (C) GSEA plot comparing differentially expressed genes upon FIP1L1 knockdown in Kasumi-1 cells with the Jaatinen Hematopoietic Stem Cell UP gene set. The heatmap displays 15 genes in the leading edge of the gene set that are most downregulated upon FIP1L1 knockdown. (D) CD34 cell surface expression measured by flow cytometry of Kasumi-1 cells 6 days after transduction with shRNAs targeting FIP1L1 or a control shRNA. Data are mean ± standard deviation (SD) of 3 independent experiments. ***P < .001, 1-way analysis of variance (ANOVA) with post hoc Tukey test. (E) Proliferation of Kasumi-1 cells after transduction with shRNAs targeting FIP1L1 or a control shRNA. Cells were seeded (day 0) after 2 days of puromycin selection. The graph displays the mean and SD of 3 technical replicates in 1 representative experiment of 2 independent experiments. **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. (F) Percentage of Annexin V–positive apoptotic Kasumi-1 cells 5 days after transduction with shRNAs targeting FIP1L1 or control shRNAs. Data are mean ± SD of 3 independent experiments. **P < .01, 1-way ANOVA with post hoc Tukey test. (G) Western blot showing FIP1L1 and actin (loading control) protein in Kasumi-1 cells after transduction with control sgRNAs targeting green fluorescent protein or sgRNAs targeting FIP1L1. (H) CD34 cell surface expression measured by flow cytometry of Kasumi-1 cells 10 days after transduction with sgRNAs targeting FIP1L1 or control sgRNAs. Data are mean ± SD of 3 independent experiments. **P < .01, Student t test. (I) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of various myeloid differentiation genes in Kasumi-1 cells transduced with sgRNAs targeting FIP1L1 or control sgRNAs. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. (J) RT-qPCR analysis of various HSC signature genes in Kasumi-1 cells transduced with sgRNAs targeting FIP1L1 or control sgRNAs. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. FDR, false discovery rate; MFI, mean fluorescent intensity; NES, normalized enrichment score.

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