Figure 4.
PIWIL4 binds to cancer-associated protein coding RNA in AML cells and its depletion induces deregulation of LSC and DNA repair–associated pathways. (A) Genomic distribution of PIWIL4 bound RNA in THP-1 cells from 2 independent PAR-CLIP experiments. (B) The top 2 significantly enriched motifs of PIWIL4-binding sites in THP-1 cells. (C) MSigDB pathway analysis of RNA targets of PIWIL4 annotated to protein-coding genic regions. (D) Heatmap of differentially expressed genes in shPIWIL4 vs scrambled transduced THP-1 cells, depicting expression of LSC-associated genes identified in HSC/CMP/GMP lineage LSCs from MLL-AF9, MOZ-TIF2, and MN1 models. (E) Cell radar analysis of differentially expressed genes. (F) gene set enrichment analysis (GSEA) of RNA-seq of PIWIL4-depleted THP-1 cells.39 (G) γ-H2AX foci in Piwil4-depleted ckit+ MLL-AF9+ cells. The horizontal size bar represents a distance of 20 μM. The right panel shows the mean percentage of cells with more than 5 foci combined from 3 independent experiments (H) Neutral comet assay depicting damaged DNA in PIWIL4-depleted cells from patients with primary AML. The size bar depicts 100 μm distance. The right panel shows a summary of the tail moment of single cells combined from 3 biological replicates. (I) Left panel: representative western blot for PIWIL4, p-ATR, p-RPA2, and γ-H2AX and β-actin as a control on PIWIL4-depleted THP-1 cells and depleted cells rescued by ectopic expression of PIWIL4. Right panel: densitometry data analyzed using ImageJ of PIWIL4, p-ATR, p-RPA2, and γ-H2AX protein levels normalized to β-actin protein levels in scrambled vs shPIWIL4-A-transduced THP-1 cells with (+) or without (−) the ectopic expression of PIWIL4. The data is a summary of 3 independent experimental replicates. (J) Native bromodeoxyuridine (BrdU) staining of PIWIL4-depleted AML cells. The left panel shows a representative figure of anti–BrdU-488–stained THP-1 cells. The size bar represents a distance of 20 μM. The right panel shows mean fluorescent intensity of anti–BrdU-488–stained cells.

PIWIL4 binds to cancer-associated protein coding RNA in AML cells and its depletion induces deregulation of LSC and DNA repair–associated pathways. (A) Genomic distribution of PIWIL4 bound RNA in THP-1 cells from 2 independent PAR-CLIP experiments. (B) The top 2 significantly enriched motifs of PIWIL4-binding sites in THP-1 cells. (C) MSigDB pathway analysis of RNA targets of PIWIL4 annotated to protein-coding genic regions. (D) Heatmap of differentially expressed genes in shPIWIL4 vs scrambled transduced THP-1 cells, depicting expression of LSC-associated genes identified in HSC/CMP/GMP lineage LSCs from MLL-AF9, MOZ-TIF2, and MN1 models. (E) Cell radar analysis of differentially expressed genes. (F) gene set enrichment analysis (GSEA) of RNA-seq of PIWIL4-depleted THP-1 cells.39 (G) γ-H2AX foci in Piwil4-depleted ckit+ MLL-AF9+ cells. The horizontal size bar represents a distance of 20 μM. The right panel shows the mean percentage of cells with more than 5 foci combined from 3 independent experiments (H) Neutral comet assay depicting damaged DNA in PIWIL4-depleted cells from patients with primary AML. The size bar depicts 100 μm distance. The right panel shows a summary of the tail moment of single cells combined from 3 biological replicates. (I) Left panel: representative western blot for PIWIL4, p-ATR, p-RPA2, and γ-H2AX and β-actin as a control on PIWIL4-depleted THP-1 cells and depleted cells rescued by ectopic expression of PIWIL4. Right panel: densitometry data analyzed using ImageJ of PIWIL4, p-ATR, p-RPA2, and γ-H2AX protein levels normalized to β-actin protein levels in scrambled vs shPIWIL4-A-transduced THP-1 cells with (+) or without (−) the ectopic expression of PIWIL4. The data is a summary of 3 independent experimental replicates. (J) Native bromodeoxyuridine (BrdU) staining of PIWIL4-depleted AML cells. The left panel shows a representative figure of anti–BrdU-488–stained THP-1 cells. The size bar represents a distance of 20 μM. The right panel shows mean fluorescent intensity of anti–BrdU-488–stained cells.

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