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

Journal: JCI Insight

Article Title: Targeting the CALR interactome in myeloproliferative neoplasms

doi: 10.1172/jci.insight.122703

Figure Lengend Snippet: Volcano plots showing the degrees of enrichment (ratio of LFQ protein intensities) in MPL-WT-Ba/F3 cells expressing (A) DEL CALR compared with WT CALR cells and (B) INS CALR compared with WT CALR cells. The x axis depicts the fold change in protein levels and the y axis the –log10 P value. For each, 3 independent experiments were performed, and the data were analyzed with MaxQuant. Changes in partner’s abundance are shown for each condition. Normalized LFQ intensities of a subset of candidate proteins to the MBP LFQ of each condition, for proteins that are (C) enriched (Hspa5/BiP, Myl9, Hspa9/Gp75, and Fli1) or (D) lost (Pdia3/Erp57 and Msi2) in mutant cells compared with WT CALR–expressing cells. (E) Confirmation of differential binding between WT CALR and mutant proteins with a subset of targets by co-immunoprecipitation (Hspa5/BiP, Myl9, Hspa9/Grp75, Fli1 and Pdia3/Erp75) in MPL-WT-Ba/F3 cells. MBP was used as an immunoprecipitation efficacy control. In C and D, mean values ± SEM are represented. Statistical significance was assessed using 1-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001. Blots are representative of 3 independent experiments.

Article Snippet: Ba/F3 cells were transduced as previously described ( 30 ) with retroviruses expressing puromycin and shRNA- Erp57 , shRNA- Fli1 (OriGene), or shRNA- Renilla as control.

Techniques: Expressing, Mutagenesis, Binding Assay, Immunoprecipitation, Control

Journal: JCI Insight

Article Title: Targeting the CALR interactome in myeloproliferative neoplasms

doi: 10.1172/jci.insight.122703

Figure Lengend Snippet: (A) Cytoplasm/membrane extracts and (B) nucleus/chromatin extracts from EV or WT CALR–, DEL CALR–, and INS CALR–expressing MPL-WT-Ba/F3 cells were used to detect, by Western blot analysis, cellular localization of CALR (MBP) and its partners: BiP, Myl9, Fli1, and Erp57. L, light exposure time; D, dark exposure time. (C) Cytoplasm (Cyto) and nuclear (Nuc) extracts from mononuclear cells isolated from healthy donors (CTRL) or patients with CALR-mutated MPN. (D) Quantitative reverse transcription PCR (qRT-PCR) analysis of MPL expression in MPL-WT-Ba/F3 cells expressing WT, DEL, or INS CALR compared with EV control. qRT-PCR analysis of ChIP assays showing (E) Fli1, (F) Erp57, and (G) CALR binding to the Mpl promoter in MPL-WT-Ba/F3 cells expressing WT, DEL, or INS CALR. Data are expressed as the percentage of pre-immunoprecipitation input for each sample and are representative of at least 3 independent immunoprecipitations. (H) qPCR analysis of Mpl in MPL-WT-Ba/F3 cells expressing EV or WT, DEL, or INS CALR transduced by Fli1 or Erp57 shRNA compared with Renilla controls (CTRL). (I) Signal distribution of Fli1 ChIP-seq peaks’ intensity in EV or WT CALR–, DEL CALR–, or INS CALR–expressing cells. (J) Mean peak intensity of Fli1 peaks per the following genomic regions: 3′-UTR, 5′-UTR, promoter TSSs, and TSSs. TSS, transcription start site for MPL-WT-Ba/F3 cells expressing EV or WT, DEL, or INS CALR. In D–H, mean values ± SEM are represented. Statistical significance was assessed using 1-way ANOVA. *P < 0.05, **P < 0.01, and ****P < 0.0001. Blots are representative of 3 independent experiments. Gapdh or histone H3 was used as a loading control. Gapdh was used as a housekeeping gene (n = 3 in triplicate).

Article Snippet: Ba/F3 cells were transduced as previously described ( 30 ) with retroviruses expressing puromycin and shRNA- Erp57 , shRNA- Fli1 (OriGene), or shRNA- Renilla as control.

Techniques: Membrane, Expressing, Western Blot, Isolation, Reverse Transcription, Quantitative RT-PCR, Control, Binding Assay, Immunoprecipitation, shRNA, ChIP-sequencing

Journal: JCI Insight

Article Title: Targeting the CALR interactome in myeloproliferative neoplasms

doi: 10.1172/jci.insight.122703

Figure Lengend Snippet: (A) Proliferation assays of MPL-WT-Ba/F3 cells expressing CALR mutant constructs treated with the C-Term peptide in the absence of IL-3 compared with untreated controls (Unt). Growth curves are means (in total number of viable cells) ± SEM (n = 6 in triplicate). (B) JAK/STAT axis analysis by Western blot analysis of MPL-WT-Ba/F3-cells expressing DEL or INS CALR treated with the C-Term peptide for different incubation times (0–2 hours). (C) Percentage (%) of cleaved caspase-3–positive MPL-WT-Ba/F3 cells expressing CALR mutants after treatment with the C-Term peptide for 4 hours (n = 3 in triplicate). In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. Dots outside of box plots represent outliers. (D) Quantification of change in MPL mean fluorescence index (MFI) in MPL-WT-Ba/F3 cells expressing DEL or INS CALR treated with the C-Term peptide for the indicated time points. (E) Co-immunoprecipitation analysis and (F) quantitative densitometry (AU) of MPL protein expression in 293T cells cotransfected with MPL-WT and MBP-tagged CALR-mutant constructs treated with the peptide for 15 minutes or 1 hour. (G) Western blot analysis of MBP expression in 293T cells transfected with MPL-WT and MBP-tagged CALR-mutant constructs and treated with the C-Term peptide for 1 hour. (H) Cytoplasm/membrane and nucleus/chromatin extracts from EV or WT, DEL, or INS CALR–expressing MPL-WT-Ba/F3 cells were used to detect, by Western blot, cellular localization of CALR partners BiP, Myl9, Fli1, and Erp57 after C-Term treatment for 1 hour. In A, C, D, and F, mean values ± SEM are represented. Statistical significance was assessed using 2-way ANOVA, ****P < 0.0001. Blots are representative of 3 independent experiments. Gapdh, histone H3, and vinculin were used as loading controls.

Article Snippet: Ba/F3 cells were transduced as previously described ( 30 ) with retroviruses expressing puromycin and shRNA- Erp57 , shRNA- Fli1 (OriGene), or shRNA- Renilla as control.

Techniques: Expressing, Mutagenesis, Construct, Western Blot, Incubation, Whisker Assay, Fluorescence, Immunoprecipitation, Transfection, Membrane