BCAA transporters in the progression of leukemia. (A) Left heat map: amino acids in the MLL-AF9 control and MLL-AF9-Gprc5c-OE cells. Log2 transformed the mean value of metabolite abundances per experiment and z-score centering per metabolite for comparison between experiments. Three independent experiments were performed with >2 replicates per experiment; n = 10. Right heat map: amino acids in MLL-AF9-WT and MLL-AF9-Gprc5c-KO. Log2-transformed mean value of metabolite abundances per experiment and z-score centering per metabolite for comparison between experiments. Two independent experiments were performed with >3 replicates per experiment; n = 7 to 11. (B) Venn diagram representing amino acids significantly upregulated in MLL-AF9-Gprc5c-OE (blue circle) or significantly downregulated in MLL-AF9-Gprc5c-KO (red circle). (C) Venn diagram representing amino acids imported by SLC7A5 or SLC43A1. (D) Schematic representation of the experimental design to rescue MLL-AF9-Gprc5c-KO cells by supplementation with BCAAs. Annexin V staining of BCAA-treated and control leukemia cells; n = 8; schematic representation of the experimental design to assess the impact of the loss of Slc7a5 or Slc43a1 in MLL-AF9. (E) Schematic representation of the experimental design to knockdown Slc7a5 and Slc43a1 in MLL-AF9 to determine impact on cell survival. (F) Quantitative (qPCR) validation of the knockdown of Slc7a5 or Slc43a1 in MLL-AF9 cells. Normalized to the housekeeping genes Oaz1/B2m and shRenilla; n = 4 to 8. (G) Heat map representing medium RNA expression from qPCR data (normalized to housekeeping gene-Oaz1/B2m and shRenilla); n = 9. (H) Annexin V staining of Slc7a5 or Slc43a1 knockdown in MLL-AF9 cells; n = 3 to 5. (I) Caspase-3 staining to determine the frequency of dead cells from Slc7a5 or Slc43a1 knockdown MLL-AF9 cells; n = 3. All represented by mean ± standard deviation. (A) Unpaired t test; (D,F-I) 2-way analysis of variance. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001;∗∗∗∗ P <0.0001. For all experiments, at least 2 independent experiments were performed.

BCAA transporters in the progression of leukemia. (A) Left heat map: amino acids in the MLL-AF9 control and MLL-AF9-Gprc5c-OE cells. Log2 transformed the mean value of metabolite abundances per experiment and z-score centering per metabolite for comparison between experiments. Three independent experiments were performed with >2 replicates per experiment; n = 10. Right heat map: amino acids in MLL-AF9-WT and MLL-AF9-Gprc5c-KO. Log2-transformed mean value of metabolite abundances per experiment and z-score centering per metabolite for comparison between experiments. Two independent experiments were performed with >3 replicates per experiment; n = 7 to 11. (B) Venn diagram representing amino acids significantly upregulated in MLL-AF9-Gprc5c-OE (blue circle) or significantly downregulated in MLL-AF9-Gprc5c-KO (red circle). (C) Venn diagram representing amino acids imported by SLC7A5 or SLC43A1. (D) Schematic representation of the experimental design to rescue MLL-AF9-Gprc5c-KO cells by supplementation with BCAAs. Annexin V staining of BCAA-treated and control leukemia cells; n = 8; schematic representation of the experimental design to assess the impact of the loss of Slc7a5 or Slc43a1 in MLL-AF9. (E) Schematic representation of the experimental design to knockdown Slc7a5 and Slc43a1 in MLL-AF9 to determine impact on cell survival. (F) Quantitative (qPCR) validation of the knockdown of Slc7a5 or Slc43a1 in MLL-AF9 cells. Normalized to the housekeeping genes Oaz1/B2m and shRenilla; n = 4 to 8. (G) Heat map representing medium RNA expression from qPCR data (normalized to housekeeping gene-Oaz1/B2m and shRenilla); n = 9. (H) Annexin V staining of Slc7a5 or Slc43a1 knockdown in MLL-AF9 cells; n = 3 to 5. (I) Caspase-3 staining to determine the frequency of dead cells from Slc7a5 or Slc43a1 knockdown MLL-AF9 cells; n = 3. All represented by mean ± standard deviation. (A) Unpaired t test; (D,F-I) 2-way analysis of variance. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001;∗∗∗∗ P <0.0001. For all experiments, at least 2 independent experiments were performed.

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