Figure 5.
FIP1L1 expression correlates to stemness gene signatures and differentiation across AML. (A) Box and whisker plot depicting the FIP1L1 expression of all patients in the AML patient cohort of The Cancer Genome Atlas (TCGA) . Differential gene expression analysis was performed comparing the highest (red) and lowest (blue) FIP1L1 expressing patients. (B) Heatmap depicting significantly enriched gene ontology (GO) terms of upregulated genes in FIP1L1 low-expressing patients and in Kasumi-1 cells upon FIP1L1 knockdown. (C) Plots from GSEA of the RNA-sequencing analysis described in panel A. (D) Western blot showing FIP1L1 and actin (loading control) protein in HL-60 and NB4 cells after transduction with control shRNAs or shRNAs targeting FIP1L1 [shRNA (2) or (3)]. (E) Percentage of CD11b+ HL-60 and NB4 cells, measured by flow cytometry, 5 days after transduction with shRNAs targeting FIP1L1 or a control shRNA. Data are mean ± standard deviation (SD) of 3 independent experiments. *P < .05, **P < .01, ***P < .001, 1-way analysis of variance (ANOVA) with a post hoc Tukey test. (F) Wright-Giemsa staining of HL-60 cell cytospins, 5 days after transduction with shRNAs targeting FIP1L1 or a control shRNA (400×). Irregular-shaped nuclei observed in shFIP1L1 image are indicative of granulocytic differentiation compared with the spherical, smooth-edged nuclei observed in the shControl image. (G) Proliferation of HL-60 and NB4 cells after transduction with shRNAs targeting FIP1L1 or a control shRNA. Cells were seeded (day 0) after 2 days of puromycin selection. The graphs display the mean and SD of 3 technical replicates in 1 representative experiment of 2 independent experiments per cell line. **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. (H) Percentage of Annexin V–positive apoptotic HL-60 and NB4 cells 4 days after transduction with shRNAs targeting FIP1L1 or control shRNAs. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with a post hoc Tukey test. FDR, false discovery rate; NES, normalized enrichment score.

FIP1L1 expression correlates to stemness gene signatures and differentiation across AML. (A) Box and whisker plot depicting the FIP1L1 expression of all patients in the AML patient cohort of The Cancer Genome Atlas (TCGA) . Differential gene expression analysis was performed comparing the highest (red) and lowest (blue) FIP1L1 expressing patients. (B) Heatmap depicting significantly enriched gene ontology (GO) terms of upregulated genes in FIP1L1 low-expressing patients and in Kasumi-1 cells upon FIP1L1 knockdown. (C) Plots from GSEA of the RNA-sequencing analysis described in panel A. (D) Western blot showing FIP1L1 and actin (loading control) protein in HL-60 and NB4 cells after transduction with control shRNAs or shRNAs targeting FIP1L1 [shRNA (2) or (3)]. (E) Percentage of CD11b+ HL-60 and NB4 cells, measured by flow cytometry, 5 days after transduction with shRNAs targeting FIP1L1 or a control shRNA. Data are mean ± standard deviation (SD) of 3 independent experiments. *P < .05, **P < .01, ***P < .001, 1-way analysis of variance (ANOVA) with a post hoc Tukey test. (F) Wright-Giemsa staining of HL-60 cell cytospins, 5 days after transduction with shRNAs targeting FIP1L1 or a control shRNA (400×). Irregular-shaped nuclei observed in shFIP1L1 image are indicative of granulocytic differentiation compared with the spherical, smooth-edged nuclei observed in the shControl image. (G) Proliferation of HL-60 and NB4 cells after transduction with shRNAs targeting FIP1L1 or a control shRNA. Cells were seeded (day 0) after 2 days of puromycin selection. The graphs display the mean and SD of 3 technical replicates in 1 representative experiment of 2 independent experiments per cell line. **P < .01, ***P < .001, multiple Student t tests using the Holm-Šidák method. (H) Percentage of Annexin V–positive apoptotic HL-60 and NB4 cells 4 days after transduction with shRNAs targeting FIP1L1 or control shRNAs. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with a post hoc Tukey test. FDR, false discovery rate; NES, normalized enrichment score.

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