Extended Data Figure 6: BCAT1 cooperates with BCR–ABL1 in blastic transformation in vivo. | Nature

Extended Data Figure 6: BCAT1 cooperates with BCRABL1 in blastic transformation in vivo.

From: Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia

Extended Data Figure 6

a, RT–qPCR analysis of Bcat1 expression in normal LSK or Lin c-Kit+ HSPCs transduced with either the vector or Bcat1 retroviruses. The expression levels are normalized and displayed relative to the control B2m expression. ***P < 0.001. b, Normal LSK or Lin c-Kit+ HSPCs were purified from healthy bone marrow and transduced with the indicated retroviruses, and the infected cells were plated in triplicate to assess colony formation after 10 days. Error bars, s.e.m. NS, not statistically significant (P > 0.05). c, Colony-forming ability of normal HSPCs expressing BCRABL1 and Bcat1. LSK cells were purified from healthy bone marrow and transduced with either the control vector or Bcat1 together with BCRABL1 (B/A) retroviruses, and double-positive cells were plated in triplicate to assess colony formation after 10 days (plated at a density of 150 cells per well). Photomicrographs show representative colonies formed in each group. Scale bar, 500 μm. Error bars, s.e.m. ***P < 0.001. d, Chimaerism of donor-derived cells after transplantation with LSK cells expressing the indicated constructs. n = 15 for each group. *P < 0.05. e, Haematoxylin and eosin staining of liver, lung and spleen sections from mice transplanted with LSK cells expressing BCRABL1 and vector or Bcat1. White arrows indicate immature myeloid cells. Scale bars, 100 μm for ×10 images and 20 μm for ×40 images. f, Plasma α-amino acid levels in mice transplanted with LSK cells infected with BCRABL1 and the vector or Bcat1. Blood plasma fractions were prepared from peripheral blood samples, methanol-extracted and dried under a vacuum. The dried extracts were labelled with NBD-F and analysed using an HPLC equipped with a fluorescence detector. Open and closed bars indicate vector control (n = 17) and Bcat1 (n = 18) specimens, respectively. *P < 0.05, **P < 0.01. g, Representative flow cytometry plots showing lineage marker expression in leukaemia cells from mice transplanted with LSK cells infected with either the control vector or Bcat1 together with BCRABL1. Leukaemia cells were analysed for their frequency of the Lin population.

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