Fig. 4: RNA-Sequencing analysis.
a Normalized expression profiles of the top 2176 genes with the largest standard deviations across all samples. The dendrogram shows samples in all groups clustered as expected. RNA samples were taken from BM cells from WT, p53+/−, ITD/ITD; p53+/−, ITD/ITD; p53−/−, and p53−/− mice (n = 3 for each group). There were only AML in BM in these ITD/ITD; p53+/− (e.g., #1376, Fig. 2a) and ITD/ITD; p53−/− mice, while p53+/- (e.g., #1381, Fig. 2a) and p53−/− mice did not show infiltration of tumor cells in BM. Differentially expressed p53 target genes in p53+/− mice compared with ITD/ITD mice were presented in Supplementary Fig. 11. Venn diagrams show the overlap of differentially expressed genes among 3 data sets including ITD/ITD; p53+/− (b: downregulated, c: upregulated). Pink = ITD/ITD vs WT; Yellow = p53+/− vs WT; Orange = ITD/ITD; p53+/− vs WT. Genes were considered to be differentially expressed based on DESeq2 statistics applying a corrected p value threshold (false discovery rate) at 0.01. Venn diagrams showing the overlap of differentially expressed genes among three data sets including ITD/ITD; p53−/− (d: downregulated; e: upregulated). Pink = ITD/ITD vs WT; Orange = p53−/− vs WT; Yellow = ITD/ITD; p53−/− vs WT. Venn diagrams show the overlap of differentially expressed genes in 2 pairwise comparisons (ITD/ITD; p53+/− vs ITD/ITD = pink; ITD/ITD; p53−/− vs ITD/ITD = yellow) (f: downregulated, g: upregulated). h TaqMan assays confirmed the RNA-Seq data in analyzed animals. ITD/ITD: n = 3 mice, ITD/ITD; p53+/−: n = 6, ITD/ITD; p53−/−: n = 4, p53+/−: n = 4 (for Lin28a and Sowaha) or 3 (for FLT3 and Htra3), p53−/−: n = 2. i Overexpression of Htra3 inhibited apoptosis induced by TGF-ß. 32D cells were transduced by a retroviral vector [45] expressing murine Htra3, and two clones with almost 100% expression of Htra3 were selected by limited dilution. Transduced 32D cells were cultured in the presence of TGF-ß without IL-3. Results presented are the mean ± SD of at least 4 independent experiments. **p < 0.01. j Representative example of macroscopic spleen colonies (CFU-S) from wild-type irradiated recipients injected with Lin− cells from ITD/ITD donors transduced with Htra3 or control cells from ITD/ITD mice (left panel). Right panel shows CFU-S from recipients injected with Lin− cells from WT donor. Spleens were fixed in Carnoy’s solution. Note that there were around 20% transplanted cells were with Htra3 k. l Represent cytospins showing different morphology of 32D WT and 32D overexpressing Lin28a cells (32D-Lin28a) on day 2 after G-CSF treatment. About 75% 32D-Lin28a cells went to differentiation (myelocytes to segmented neutrophils [45, 46]), while only 11% 32D WT cells differentiated. Similar differences were also observed on day 4, day 6, and day 8 after G-CSF treatment (data not shown). m Cytospin from mouse #1884 with erythroid leukemia showing strong infiltration of erythroblasts in spleen (left panel). Cytospin from mouse #1993 with AML showing >50% myeloblasts in BM spleen. Most myeoloblasts showed morphological features of immature monocytes similar as leukemic cells from double-transgenic mice (Fig. 2a). Mice #1884 and #1994 were transplanted with ITD/ITD lin− cells overexpressing Htra3 and overexpressing Htra3/Lin28a knockdown, respectively.
