ckit  (Novus Biologicals)

Journal: Nature Communications

Article Title: Thrombopoietin increases susceptibility for EVI1 + KMT2A-MLLT3-driven AML expressing stem cell genes linked to poor outcome

doi: 10.1038/s41467-025-67611-w

Figure Lengend Snippet: Number of LT-HSC ( A ) and MPP1 ( B ) normalized to 10 6 Lin - BM cells in animals treated with either TPO (dark blue, n = 15), RP (light blue, n = 6), poly(I:C) (lilac, n = 5), 5FU-3d (gray, n = 4), 5FU-6d (white dots, n = 5) or PBS (white triangles, n = 14 (A) and n = 17 ( B ). Flow cytometric quantification LT-HSC ( C ) and MPP1 ( D ) expressing Evi1 high , Evi1 low and Evi1 neg treated with TPO: n = 15, RP: n = 6, pI:pC: n = 5, 5-FU 3 d: n = 4, 5-FU 6 d: n = 5, PBS: n = 13 ( E ) Representative high-resolution image of femurs of KME mice after PBS (left) or TPO (right) injection on DOX for 5 days (starting 48 h after injection). Bones from 4 mice per condition stained with anti-endomucin (white), anti-cKit (red) and anti-GFP (green). Scale bar=1000 μm. F 3-D images of Evi1 + ( = GFP + ) c-Kit + cells. Cells of interest (Evi1 + /Kit + ) annotated based on nuclear expression of Evi1-GFP (green) and membranous expression of Kit (red). G Density of GFP + Kit + cells in femurs from PBS- vs TPO-treated KME mice. Statistical significance determined using two-sided unpaired Mann–Withney test. (Ctrl, n = 4, TPO, n = 4). H Flow cytometry plots showing the fractions (%) of Evi1 high LT-HSC in the G 0 , G 1 and S/G 2 /M phases 48 h after TPO or PBS treatment. I Flow cytometric quantification of the proportion (%) of LT-HSC in the G 0 , G 1 and S/G 2 /M phases depending on Evi1 expression levels (high, low or negative) 48 h after PBS ( n = 2) or TPO ( n = 3) treatment. J Flow cytometry plots showing the fractions (%) of Evi1 high MPP1 in the G 0 , G 1 and S/G 2 /M phases 48 h after TPO or PBS treatment. K Flow cytometric quantification of the proportion (%) of MPP1 in the G 0 , G 1 and S/G 2 /M phases depending on Evi1 expression levels (high, low or negative) 48 h after PBS ( n = 2) or TPO ( n = 3) treatment. L Representative histogram showing the mean fluorescence intensity (MFI) of Mpl surface expression on LT-HSC in the different tested conditions (FMO = Fluorescence Minus One). M Flow cytometric quantification of Mpl expression in HSPC of PBS ( n = 5), and TPO ( n = 3)- treated animals. Values normalized to Lin - BM MFI (FMO = Fluorescence Minus One). N Correlation between Mpl and Evi1 expression in HSPC populations ( n = 7) based on MFI. Pearson correlation: r = 0.9174, p = 0.001323 (two-sided). O Mpl expression (MFI) in LT-HSC expressing different levels of Evi1 from TPO-treated mice normalized to LT-HSC from PBS-treated mice. (Evi1 high LT-HSC/LT-HSC MFI ratio; PBS: 1.12 ± 0.12, n = 7/4; TPO: 0.71 ± 0.07, n = 3/3, p = 0.04). N = number of mice. Statistically significant differences between indicated treatments and PBS were calculated by 1-way ANOVA ( A – B ) or 2-way ANOVA ( C – D , I , K , M , O ) followed by Sidak multiple comparison test ( M , O ), Tukey’s post-test ( C–D ) and two-sided unpaired t -test ( A–B, I , K ) and data are presented as mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Source data provided in the Source Data file.

Article Snippet: Double-positive (Evi1 + -Ckit + ) HSC cells were manually annotated using the spots module of Imaris software (v10.2 Bitplane AG, Oxford Instruments) to ensure a minimal error rate.

Techniques: Expressing, Injection, Staining, Flow Cytometry, Fluorescence, Comparison

Journal: Nature Communications

Article Title: Thrombopoietin increases susceptibility for EVI1 + KMT2A-MLLT3-driven AML expressing stem cell genes linked to poor outcome

doi: 10.1038/s41467-025-67611-w

Figure Lengend Snippet: A Number and types of colonies formed by Evi1 high LT-HSC and Evi1 high MPP1 from KME mice (on DOX) harvested 2 days after TPO (200μg/kg BW) or PBS treatment (LT-HSC (PBS): n = 5, LT-HSC (TPO): n = 4, MPP1 (PBS): n = 4, MPP1 (TPO): n = 6). More “type IV” colonies were formed by TPO-exposed cells. n = number of MC. B Representative pictures of colonies (2.5x) (top) and images of cytospin preparations (60x) (bottom) of Evi1 high LT-HSC- (left) and Evi1 high MPP1-derived-cells (right) from mice with or without TPO treatment grown for 10 days. Scale bar = 10 μm. C Proportion (%) of Evi1 + ( = GFP + ) cells in Evi1 high LT-HSC (green & brown)- and Evi1 high MPP1 (light green & blue)-derived colony-forming cells from mice with or without TPO treatment. LT-HSC: TPO ( n = 3), PBS ( n = 5); MPP1: TPO ( n = 6), PBS ( n = 5). D Experimental outline: transplantation of sorted LT-HSC or MPP1 from TPO-stimulated KME mice wildtype recipients ( E ) Kaplan–Meyer plot of disease-free mice. Transplantation of sorted HSCP from TPO-treated mice resulted in accelerated disease development: MPP1: 41 d vs 79 d, n = 14 vs n = 7, p = 0.025; LT-HSC: 46 d vs 90 d, n = 21 vs n = 11, p = 0.028; Mantel-Cox test. F Spleen-to-BW ratio and ( G ) representative pictures of spleens from diseased mice transplanted with Evi1 high LT-HSC or -MPP1 with and without TPO pre-treatment: LT-HSC-derived: TPO ( n = 20), PBS ( n = 10); MPP1-derived: TPO ( n = 13), PBS ( n = 6). Gray area in ( F ) : spleen size in healthy mice. H Proportion (%) of Evi1 + ( = GFP + ) BM cells from diseased mice transplanted with LT-HSC or MPP1 from TPO-treated or untreated controls. LT-HSC-derived: TPO ( n = 19), PBS ( n = 9); MPP1-derived: TPO ( n = 13), PBS ( n = 6). (I ) Flow cytometric quantification of the proportion (%) of Evi1 + leukemic cells in diseased animals transplanted with LT-HSC or MPP1 from TPO- or PBS-treated donors. J–K Correlation between disease latency and the proportion of Evi1 + BM cells in diseased mice transplanted with ( n = 19) ( J ) or without ( n = 23) TPO-treated ( K ) cells. Two-sided Pearson correlation test was used to calculate the significance. N = number of mice. Statistical significance was calculated with 1-way ANOVA ( C , F , H ) and 2-way ANOVA ( A ) followed by Tukey’s post-hoc were used to test for significance and data are represented as mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Source data are provided in the Source Data file.

Article Snippet: Double-positive (Evi1 + -Ckit + ) HSC cells were manually annotated using the spots module of Imaris software (v10.2 Bitplane AG, Oxford Instruments) to ensure a minimal error rate.

Techniques: Derivative Assay, Transplantation Assay

Journal: Nature Communications

Article Title: Thrombopoietin increases susceptibility for EVI1 + KMT2A-MLLT3-driven AML expressing stem cell genes linked to poor outcome

doi: 10.1038/s41467-025-67611-w

Figure Lengend Snippet: A PCA shows a marked separation of the gene expression signatures of KME AML cells by TPO exposure of the donors with only a minor influence of Evi1 expression. B AML cells emerging from TPO-treated KME BM expressed similar or higher Evi1 and Erg levels than LT-HSC, or GMP-derived iKMT2A-MLLT3 blasts reported earlier . C Venn diagram showing the number of common DEGs the different AML cell fractions (Evi1 + , Evi1 - , and bulk) emerging from TPO- vs. PBS-stimulated KME BM cells. D Volcano plot of DEGs in Evi1 + AML cells emerging from TPO- or PBS-stimulated KME BM cells. E GSEA (MSigDB) analysis of DEGs in AML cells emerging from TPO- or PBS-treated KME BM cells revealed associations to signatures of aberrant Hoxa9 activity in both, the bulk and Evi1 + cell fractions. F KEGG analysis of DEGs in AML cells (bulk) emerging from TPO- or PBS-treated KME BM cells revealed up-regulation JAK-STAT and NF-κB signaling pathways. G GSEA revealed significant upregulation of JAK-STAT (top) and MAPK (bottom) pathways related genes in bulk blasts from symptomatic mice transplanted with TPO-treated KME BM cells. H GSEA revealed significant upregulation of the Biocarta (MSig-DB) TPO pathway-related genes in bulk blasts from symptomatic mice transplanted with TPO-treated KME BM cells. p-values were calculated with quasi-likelihood F-tests from EdgeR package ( D ) or permutation test of GSEA function from clusterProfiler R package ( E–H ).

Article Snippet: Double-positive (Evi1 + -Ckit + ) HSC cells were manually annotated using the spots module of Imaris software (v10.2 Bitplane AG, Oxford Instruments) to ensure a minimal error rate.

Techniques: Gene Expression, Expressing, Derivative Assay, Activity Assay, Protein-Protein interactions

Journal: Nature Communications

Article Title: Thrombopoietin increases susceptibility for EVI1 + KMT2A-MLLT3-driven AML expressing stem cell genes linked to poor outcome

doi: 10.1038/s41467-025-67611-w

Figure Lengend Snippet: A K-means clustering according to ERG and MECOM expression levels of the AML patients from the TARGET cohort . B Scatterplot comparing DEGs of TPO- vs PBS-exposition derived iKMT2A-MLLT3 murine AML with DEGs of EVI1 high vs EVI1 intermediate patients from the TARGET cohort . C Venn diagram illustrating similarities between DEGs in the human EVI1 + AML and the mouse Evi1 + AML signatures from 4 AML patient cohorts (TARGET, BEAT, LEUCEGENE, ST.JUDE) – . Scatterplots showing correlations of selected top up-regulated genes IL12Rβ2 ( D ) and INPP4B ( E ) compared to MECOM in the AML patients of the TARGET cohort. Pearson correlations were calculated for all patients (on the top), or only in group of patients with KMT2A-r, in KMT2A-MLLT3 or without KMT2A genomic rearrangements (colored in blue, green and purple respectively). Two-sided Pearson correlation test was used to calculate the significance for each group. F Survival curves for AML patients from the TARGET cohort split according to the median gene expression (TPM) of MECOM (top), IL12Rβ2 (middle) and INPP4B (bottom). p -values were calculated with Log-rank test.

Article Snippet: Double-positive (Evi1 + -Ckit + ) HSC cells were manually annotated using the spots module of Imaris software (v10.2 Bitplane AG, Oxford Instruments) to ensure a minimal error rate.

Techniques: Expressing, Derivative Assay, Gene Expression

Journal: Nature Communications

Article Title: Thrombopoietin increases susceptibility for EVI1 + KMT2A-MLLT3-driven AML expressing stem cell genes linked to poor outcome

doi: 10.1038/s41467-025-67611-w

Figure Lengend Snippet: A Expression of selected common higher expressed DEGs in mouse and human Evi1/EVI1 high AML (MECOM, MPL, IL12Rβ2, INPP4B and HOXA9) in different human AML cells (DepMap 23Q4 Public). Note: OCI-AML4 is the only KMT2A-r cell line expressing significant levels of all genes. B RT-qPCR assessed mRNA levels of MECOM, MPL, IL12Rβ2, INPP4B and HOXA9 in OCI-AML4 virally expressing shRNA targeting MECOM, MPL, IL12Rβ2, HOXA9 and scramble control. The values represent the average of at least 2 shRNA/target (Supplementary Fig. ). Growth curves of OCI-AML4 ( C ), MOLM-13 ( F ), HL-60 (I ), THP-1 (L ) AML cells expressing the indicated shRNA. Only the statistically significant differences (based on adjusted p -value from Dunnett’s test) are marked. Number of colonies formed by OCI-AML4 ( D ), MOLM-13 ( G ), HL-60 (J ), THP-1 ( M ) AML cells expressing the indicated shRNA 14 days after plating in MC (H4534). Representative pictures of colonies formed by OCI-AML4 ( E ), MOLM-13 ( H ), HL-60 (K ), THP-1 ( N ) cells expressing the indicated shRNAs after 14 days in MC (2.5x magnification). O Number and types of colonies formed by Evi1 high iKMT2A-MLLT3 AML cells emerging from TPO-stimulated BM expressing the indicated shRNA, after 7 days in MC (M3534). P Percentage of GFP- or mCherry-positive Evi1 high iKMT2A-MLLT3 AML cells expressing Mecom, Il12rb2 or Scramble shRNA harvested from MC plates. Q Mecom and Il12rβ2 mRNA expression in Evi1 high iKMT2A-MLLT3 AML cells expressing the respective shRNA harvested from MC plates. R Representative pictures from colonies formed by Evi1 high iKMT2A-MLLT3 AML cells expressing the different shRNAs after 7 days in MC (M3534, 2.5x magnification). All experiments (except targeting of INPP4B) have been performed in 2 independent series using 2 shRNAs per target. Statistically significant differences to the Scramble controls were calculated by using 1-way ANOVA ( B , D , G , J , M , Q ) and 2-way ANOVA ( C , F , I , L , O ) followed by Dunnett’s post test to determine significance and data are represented as mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). For bar- and dot-plot figures, source data are provided in the Source Data file.

Article Snippet: Double-positive (Evi1 + -Ckit + ) HSC cells were manually annotated using the spots module of Imaris software (v10.2 Bitplane AG, Oxford Instruments) to ensure a minimal error rate.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Control