Fig. 3: TPO stimulation rapidly modulates gene expression of iKMT2A-MLLT3 HSPC.
A Experimental setup: Flt3-LSK cells were isolated 48 h after a single dose of TPO followed by 2 or 5 days on DOX to induce expression of the iKMT2A-MLLT3 fusion. RNA from Flt3- LSK cells was sequenced at the single cell level using hash-tagged oligos (HTO) and CITE-seq antibodies (ADT). B T-SNE dimensionality reduction plot, with color illustrating the annotation of the iKMT2A-MLLT3 HSPC clusters based on published transcriptomic signatures30,89,90. C T-SNE dimensionality reduction plot, with color illustrating the gating of the cells using CITE-seq markers, following the strategy of Fast et al.39. D Heatmap showing the number of down-regulated (left column) and up-regulated (right column) DEGs (FDR < 0.05) in TPO- vs PBS exposed cells after 2 days on DOX. Volcano plots of DEGs in TPO- vs PBS exposed ST-HSC_2 (E) or MPP1_2 (F) cell clusters after 2 days on DOX. Significant DEG (FDR < 0.05) are colored in red. P-values were calculated with likelihood ratio tests from EdgeR package. G MSigDB pathway analysis (FDR < 0.05) of DEGs in TPO- vs PBS-exposed HSPC after 2 days on DOX. p-values were calculated with two-sided t-test of the camera function from the limma R package. H Heatmap showing the number of down-regulated (left column) and up-regulated (right column) DEGs (FDR < 0.05) in TPO- vs PBS exposed HSPC after 5 days on DOX. Volcano plots of DEGs in TPO- vs PBS-exposed LT-HSC (I) or MPP2-3 (J) after 5 days on DOX. Significant DEG (FDR < 0.05) are colored in red. p-values were calculated with likelihood ratio tests from EdgeR package. K MSigDB pathway analysis (FDR < 0.05) of DEGs in TPO- vs PBS-exposed HSPC after 5 days on DOX. p-values were calculated with two-sided t-test of the camera function from the limma R package.
