Figure 4.
3'UTR-APA regulates AML1-ETO expression. (A) Genome browser tracks depicting normalized sequencing reads (reads per million [RPM]) in the RUNX1T1 (aka ETO) gene obtained from 3'READS of Kasumi-1 cells transduced with a control shRNA or shRNAs targeting FIP1L1 [shRNA (1) or (2)]. The full RUNX1T1 genomic structure is shown (top) with the purple, boxed region expanded (below). The percent usage of each indicated PAS was calculated by using 3'READS and is shown in the bar graph to the right. n = 3, shControl and shFIP1L1 (2); n = 4 shFIP1L1 (1). (B) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of AML1-ETO 3'UTR length in Kasumi-1 cells transduced with a third, unique shRNA (3) targeting FIP1L1 or a control shRNA. Usage of the PAS at 3.7 kb was measured relative to total AML1-ETO mRNA using primer pairs upstream of the 3.7 kb PAS and 1 kb PAS, respectively. Data are mean ± standard deviation (SD) of 3 independent experiments. **P < .01, Student t test. (C) Western blot showing FIP1L1 and actin (loading control) protein in 293T cells transfected with a FLAG-tagged, doxycycline (Dox)-inducible FIP1L1 overexpression construct, with and without 1 μg/mL Dox in the culture medium for 24 hours. The black arrow indicates endogenous FIP1L1. The red arrow indicates FLAG-tagged FIP1L1. (D) Genome browser tracks depicting normalized sequencing reads (RPM) in the RUNX1T1 (aka ETO) gene obtained from 3'READS of 293T cells transfected with a Dox-inducible FIP1L1 overexpression construct, with and without 1 μg/mL Dox in the culture medium for 24 hours. The full RUNX1T1 genomic structure is shown (top) with the purple, boxed region expanded (below). (E) Western blot showing FIP1L1, AML1-ETO, and tubulin (loading control) protein in Kasumi-1 cells after transduction with control shRNAs or shRNAs targeting FIP1L1. AML1-ETO protein was quantified by normalizing AML1-ETO signal intensity to tubulin signal intensity using LI-COR Image Studio software. Normalized protein quantifications from 3 independent experiments are shown in the bar graph below. Data are mean ±SD. **P < .01, ***P < .001, one-way analysis of variance (ANOVA) with a post hoc Tukey test. (F) Plots from GSEA of the RNA-sequencing experiment shown in Figure 3A (supplemental Figure 5A-B). The top plot displays genes that are downregulated by AML1-ETO; the bottom plot displays those that are upregulated by AML1-ETO. (G) Relative ratio of Renilla to firefly luciferase in Kasumi-1 cells nucleofected with the indicated dual luciferase reporter construct containing variable RUNX1T1 (aka ETO) 3'UTRs. Data are mean ± SD of 4 independent experiments. ***P < .001, 1-way ANOVA with a post hoc Tukey test. FDR, false discovery rate; NES, normalized enrichment score.

3'UTR-APA regulates AML1-ETO expression. (A) Genome browser tracks depicting normalized sequencing reads (reads per million [RPM]) in the RUNX1T1 (aka ETO) gene obtained from 3'READS of Kasumi-1 cells transduced with a control shRNA or shRNAs targeting FIP1L1 [shRNA (1) or (2)]. The full RUNX1T1 genomic structure is shown (top) with the purple, boxed region expanded (below). The percent usage of each indicated PAS was calculated by using 3'READS and is shown in the bar graph to the right. n = 3, shControl and shFIP1L1 (2); n = 4 shFIP1L1 (1). (B) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of AML1-ETO 3'UTR length in Kasumi-1 cells transduced with a third, unique shRNA (3) targeting FIP1L1 or a control shRNA. Usage of the PAS at 3.7 kb was measured relative to total AML1-ETO mRNA using primer pairs upstream of the 3.7 kb PAS and 1 kb PAS, respectively. Data are mean ± standard deviation (SD) of 3 independent experiments. **P < .01, Student t test. (C) Western blot showing FIP1L1 and actin (loading control) protein in 293T cells transfected with a FLAG-tagged, doxycycline (Dox)-inducible FIP1L1 overexpression construct, with and without 1 μg/mL Dox in the culture medium for 24 hours. The black arrow indicates endogenous FIP1L1. The red arrow indicates FLAG-tagged FIP1L1. (D) Genome browser tracks depicting normalized sequencing reads (RPM) in the RUNX1T1 (aka ETO) gene obtained from 3'READS of 293T cells transfected with a Dox-inducible FIP1L1 overexpression construct, with and without 1 μg/mL Dox in the culture medium for 24 hours. The full RUNX1T1 genomic structure is shown (top) with the purple, boxed region expanded (below). (E) Western blot showing FIP1L1, AML1-ETO, and tubulin (loading control) protein in Kasumi-1 cells after transduction with control shRNAs or shRNAs targeting FIP1L1. AML1-ETO protein was quantified by normalizing AML1-ETO signal intensity to tubulin signal intensity using LI-COR Image Studio software. Normalized protein quantifications from 3 independent experiments are shown in the bar graph below. Data are mean ±SD. **P < .01, ***P < .001, one-way analysis of variance (ANOVA) with a post hoc Tukey test. (F) Plots from GSEA of the RNA-sequencing experiment shown in Figure 3A (supplemental Figure 5A-B). The top plot displays genes that are downregulated by AML1-ETO; the bottom plot displays those that are upregulated by AML1-ETO. (G) Relative ratio of Renilla to firefly luciferase in Kasumi-1 cells nucleofected with the indicated dual luciferase reporter construct containing variable RUNX1T1 (aka ETO) 3'UTRs. Data are mean ± SD of 4 independent experiments. ***P < .001, 1-way ANOVA with a post hoc Tukey test. FDR, false discovery rate; NES, normalized enrichment score.

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