2 6 1 3 1 1 1 8 1 0 2 1 1 9 1 4 1 7 1 5 s epidermitidis atcc  (ATCC)

Journal: Scientific reports

Article Title: FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

doi: 10.1038/s41598-024-64829-4

Figure Lengend Snippet: Figure 3. DNA methylation analysis of FPDMM-mimicking HPCs. (a) Overlaps of hypermethylated (left) and hypomethylated (right) CpGs between RUNX1WT/R201Q (blue) and RUNX1WT/Y287X (red) HPCs. (b) (Left) The known HOCOMOCO v11 binding motif for GABPA (top), FEV (middle), and ELF1 (bottom). (Middle and right) Distribution of ETS family TF-binding motif-enrichment. The X- and Y-axes show the distance from DMC (bp) and probability of TF-binding motifs, respectively. Solid lines are probabilities at ± 5 kb for hypermethylated DMCs in RUNX1WT/R201Q (middle) and RUNX1WT/Y287X (right) HPCs, and dashed lines are probabilities at ± 5 kb from randomly selected CpGs. Data that are significantly enriched (E-value of the Fisher’s exact test < 0.05) and ranked in the top-20 of Fisher E-value rankings are presented. Blue: GABPA- binding motif, red: FEV-binding motif, and pink: ELF1-binding motif. (c) Expression of ELF1 (left) and FLI1 (right) in RUNX1WT/R201Q (blue) and RUNX1WT/Y287X (red) HPCs compared to wild-type cells, confirmed using qRT-PCR. The X-axis shows the target genes, and the Y-axis represents the fold-change. Data are presented as the mean ± SD of four biological replicates. Asterisks denote significant difference: *P < 0.05 and **P < 0.01. (d) Distribution of enrichment for FLI1-binding sites, as determined by CUT&RUN sequencing for FLI1 in wild- type HPCs, showing the regions within ± 5 kb of the hypermethylated DMCs in RUNX1WT/Y287X HPCs (red line). Gray line represents FLI1-binding site-enrichment at regions around randomly selected CpGs.

Article Snippet: Primary antibody against human FLI1 (FLI1 (D7N5M) Rabbit mAb; #35980; Cell Signaling Technology) was added and cells were incubated overnight at 4 °C.

Techniques: DNA Methylation Assay, Binding Assay, Expressing, Quantitative RT-PCR, Sequencing

Journal: Scientific reports

Article Title: FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

doi: 10.1038/s41598-024-64829-4

Figure Lengend Snippet: Figure 4. Identification of site-specificity of DNA demethylation in FLI1. (a) Confirmation of the DOX- inducible expression of FLI1 in DOX-treated iPSCs (plus DOX) compared with untreated iPSCs (minus DOX) by qRT-PCR. (b) (Top) The known HOCOMOCO v11 binding motif for FLI1. S: G/C, R: A/G, and M: A/C. (Bottom) Distribution of FLI1-binding motif-enrichment. The solid line represents the probability at ± 5 kb from demethylated CpGs in FLI1-overexpressing iPSCs, and the dashed line represents the probability at ± 5 kb from randomly selected CpGs.

Article Snippet: Primary antibody against human FLI1 (FLI1 (D7N5M) Rabbit mAb; #35980; Cell Signaling Technology) was added and cells were incubated overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay

Journal: Scientific reports

Article Title: FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

doi: 10.1038/s41598-024-64829-4

Figure Lengend Snippet: Figure 5. Effect of FLI1 downregulation on megakaryocytic differentiation. (a) Confirmation of the expression of FLI1 in negative-control-knockdown (nc-KD, gray) and FLI1-knockdown (FLI1-KD, yellow) HPCs compared with that in wild-type HPCs using qRT-PCR. The X-axis indicates the target genes, and the Y-axis indicates the fold-change. Data are presented as the mean ± SD of three biological replicates. Asterisks denote significant difference: *P < 0.05. (b) Representative plot for flow cytometric analysis of CD41+CD42b+ Mks per 20,000 negative-control-knockdown and FLI1-knockdown HPCs. (c) Percentages of CD41+CD42b+ Mks per 20,000 negative-control-knockdown (nc-KD, gray) and FLI1-knockdown (FLI1-KD, yellow) HPCs. Data are presented as the mean ± SD of three biological replicates. Asterisks denote significant difference: *P < 0.05.

Article Snippet: Primary antibody against human FLI1 (FLI1 (D7N5M) Rabbit mAb; #35980; Cell Signaling Technology) was added and cells were incubated overnight at 4 °C.

Techniques: Expressing, Negative Control, Knockdown, Quantitative RT-PCR

Journal: Scientific reports

Article Title: FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

doi: 10.1038/s41598-024-64829-4

Figure Lengend Snippet: Figure 6. Rescue of deficient megakaryocytic differentiation in FPDMM-mimicking HPCs by FLI1 overexpression. (a) Schematic representation of the megakaryocytic differentiation method. FPDMM- mimicking iPSCs were transfected with a lentiviral vector containing the DOX-inducible FLI1 expression system. (b) Confirmation of the expression of FLI1 in Y287X-mock (red) and Y287X-FLI1 (orange) HPCs using qRT-PCR. Data are presented as the mean ± SD of six biological replicates. The asterisk denotes significant difference: *P < 0.05. (c) Percentages of CD41+CD42b+ Mks per 20,000 wild-type (WT, gray), Y287X-mock (red), and Y287X-FLI1 (orange) HPCs. Data are presented as the mean ± SD of six biological replicates. The asterisk denotes significant difference: **P < 0.01 and ns, not significant.

Article Snippet: Primary antibody against human FLI1 (FLI1 (D7N5M) Rabbit mAb; #35980; Cell Signaling Technology) was added and cells were incubated overnight at 4 °C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: Scientific reports

Article Title: FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

doi: 10.1038/s41598-024-64829-4

Figure Lengend Snippet: Figure 7. Induction of DNA hypomethylation in FPDMM-mimicking HPCs by FLI1 overexpression. (a) Scatter plot showing the percent methylation scores of 1344 CpG sites between Y287X-mock and Y287X- FLI1 HPCs. The X- and Y-axes indicate percent methylation scores for Y287X-mock and Y287X-FLI1 HPCs, respectively. The solid line represents equal percent methylation scores between samples, and dashed lines represent differences in percent methylation scores of > 25%. (b) Percentage point distributions of 1344 CpG sites between Y287X-FLI1 and Y287X-mock HPCs (FLI1-mock, orange) and of the same number of CpG sites randomly selected among CpG sites covered by sequencing (random, gray). Data are presented as the mean ± SD. The asterisk denotes significant difference: ***P < 0.001. (c) Distributions of enrichment for FLI1- binding sites, as determined by CUT&RUN sequencing for FLI1 in Y287X-mock (left) and Y287X-FLI1 HPCs (right), showing the regions within ± 5 kb of the hypermethylated DMCs in RUNX1WT/Y287X HPCs (red lines). Gray lines represent FLI1-binding site-enrichments at regions around randomly selected CpGs.

Article Snippet: Primary antibody against human FLI1 (FLI1 (D7N5M) Rabbit mAb; #35980; Cell Signaling Technology) was added and cells were incubated overnight at 4 °C.

Techniques: Over Expression, Methylation, Sequencing, Binding Assay