Extended Data Figure 5: Roles of Bcat1 in differentiation, proliferation and leukaemia development in vivo. | Nature

Extended Data Figure 5: Roles of Bcat1 in differentiation, proliferation and leukaemia development in vivo.

From: Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia

Extended Data Figure 5

a, RT–qPCR analysis of Bcat1 expression. Lin cells from NUP98–HOXA9/BCR–ABL-induced BC-CML were infected with shCtrl or Bcat1 shRNA (shBcat1-a and shBcat1-b) for three days and resorted for analysis of Bcat1 expression. The expression levels are normalized to the level of B2m expression and displayed relative to the control, which was arbitrarily set at 1. Error bars, s.e.m. of triplicate PCRs. **P < 0.01. b, RT–qPCR analysis of Bcat1 expression in leukaemia cells isolated from diseased mice transplanted with shCtrl- or shBcat1-expressing BC-CML cells. The expression levels are normalized and displayed relative to the B2m control. ***P < 0.001. c, Bcat2 expression in shBcat1-expressing cells. Lin cells from NUP98–HOXA9/BCR–ABL-induced BC-CML were infected with shCtrl or Bcat1 shRNA (shBcat1-a and shBcat1-b) for three days and resorted for analysis of Bcat2 expression. The expression levels are normalized to the level of B2m and are displayed relative to the control arbitrarily set at 1. Error bars, s.e.m. of triplicate PCRs. NS, not statistically significant (P > 0.05). d, Functional rescue of the shBcat1-induced reduction in colony-forming ability with the expression of shRNA-resistant mutant Bcat1 cDNA. Primary Lin BC-CML cells transduced with the vector or shRNA-resistant Bcat1 gene together with the indicated shRNA constructs. **P < 0.01 compared with the vector and shBcat1-b. e, f, Colony-forming ability of primary HSPCs. e, Normal HSPCs purified from bone marrow on the basis of their LSK phenotype were transduced with the Bcat1 shRNAs (shBcat1-a and shBcat1-b) and plated for colony formation. NS, not statistically significant (P > 0.05). f, Normal HSPCs were plated for colony formation with the indicated concentrations of gabapentin or PBS (−). NS, not statistically significant (P > 0.05). **P < 0.01 compared with the PBS control. Photomicrographs showing representative colonies formed under each condition. Scale bars, 500 μm. Three hundred LSK cells were plated per well in triplicate for the evaluation of colony-forming activity. Error bars, s.e.m. g, Haematoxylin and eosin staining of sections of the liver, lung and spleen at the time of onset of clinical signs (top six rows) and of tissue sections from a disease-free survivor (bottom two rows). White arrows indicate immature myeloid cells. Portal triad (PT), haemorrhagic necrosis (N), central veins (CV), arteriolar profiles (A), bile ducts (B), veins (V), white pulp (WP) and red pulp (RP) are indicated. Scale bars, 100 μm for images at ×10 and 20 μm for images at ×40 magnification. h, Representative flow cytometry plots showing lineage marker expression in leukaemia cells from mice transplanted with the shRNA-infected BC-CML cells. Leukaemia cells were analysed for their frequency of the Lin population. ik, Effect of conditional Bcat1 knockdown on BC-CML maintenance in vivo. i, Lin BC-CML cells were infected with doxycycline-inducible shRNAs against shBcat1-b or a control (shCtrl) and transplanted into recipients (1,500 cells per recipient). After 10 days of the transplantation with leukaemia cells expressing the indicated constructs, (j) donor-derived chimaerisms were analysed. Mice were then fed with chow containing doxycycline to induce shRNA expression, and (k) survival was monitored. The data shown are from two independent experiments. n = 4 for shCtrl with no Dox (DOX); n = 7 for shBcat1-b, DOX; and n = 9 each for shCtrl with Dox (DOX+) and shBcat1-b, DOX+. **P < 0.01 (shCtrl versus shBcat1-b, DOX+). NS, not statistically significant (P > 0.05). l, Cell cycle distribution of the shRNA-infected leukaemia cells. Live leukaemia cells were isolated from mice transplanted with shRNA-infected BC-CML cells, fixed and stained with propidium iodide for analysis of cell cycle distribution via flow cytometry. m, Apoptotic cells from leukaemic mice transplanted with shRNA-infected BC-CML cells were analysed via flow cytometry using Annexin V and 7-aminoactinomycin D (7-AAD) staining.

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