Figure 3.
SAM rescues the depletion of methionine in an ROS-independent manner. (A) Schematic illustration of the methionine cycle and associated pathways. Homocysteine can be recycled back to methionine via the methyltransferase activity of MTR and/or BMHT/BHMT2. (B) Relative viable cell growth of HL60 cells grown for 48 hours in the absence of methionine and increasing concentrations of SAM and S-(5′-adenosyl)-L-methionine p-toluenesulfonate salt (SAM-pTSA) (n = 3). (C) Relative viable cell growth of HL60 cells grown for 6 days in the absence of methionine and increasing concentrations of SAM and SAM-pTSA. (D-E) Relative viable cell growth of HL60 and MOLM13 cells grown for 48 hours in the absence of methionine and supplemented with glutathione (GSH) and taurine (n = 3). (F-G) Relative viable cell growth of HL60 and MOLM13 cells grown for 48 hours in the absence of methionine and supplemented with dimethyl-α ketoglutarate (DiM-αKG) vitamin B12 and folic acid (n = 3). (H) ROS levels in the AML cell lines HL60, MOLM13, and K562 as determined by the total cellular ROS probes CellROX and DCF-DA and the superoxide probe MitoSOX.Buthionine sulfoximine (BSO) was used as positive control for CellROX and dichlorodihydrofluorescein diacetate (DCF-DA) and phorbol 12-myristate-13-acetate (PMA) and menadione sodium bisulfite (MSB) for MitoSOX. (I) Relative cell counts of MOLM13 cells grown in the presence or absence of methionine and N-acetylcysteine (NAC) for 24 and 48 hours (n = 4). (J) Absolute cell counts of MOLM13 cells grown with and without cysteine and in the absence or presence of 0.1 mM or 2 mM NAC for 24 hours (n = 4). (K) Graph and dotplot of ROS (DCF-DA) MFI in MOLM13 cells depleted of cysteine alone or also supplemented with 2 mM NAC (representative example from n = 3 experiments). (L) Bar graph and histogram of ROS (DCF-DA) MFI in MV4-11 cells depleted of cysteine alone or also supplemented with 2 mM NAC (representative example of n=3 experiments). Histogram representative of the 3 replicates. All experiments: error bars represent mean ± SEM. Statistical analysis by Student t test or ordinary 1-way analysis of variance (ANOVA), ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.

SAM rescues the depletion of methionine in an ROS-independent manner. (A) Schematic illustration of the methionine cycle and associated pathways. Homocysteine can be recycled back to methionine via the methyltransferase activity of MTR and/or BMHT/BHMT2. (B) Relative viable cell growth of HL60 cells grown for 48 hours in the absence of methionine and increasing concentrations of SAM and S-(5′-adenosyl)-L-methionine p-toluenesulfonate salt (SAM-pTSA) (n = 3). (C) Relative viable cell growth of HL60 cells grown for 6 days in the absence of methionine and increasing concentrations of SAM and SAM-pTSA. (D-E) Relative viable cell growth of HL60 and MOLM13 cells grown for 48 hours in the absence of methionine and supplemented with glutathione (GSH) and taurine (n = 3). (F-G) Relative viable cell growth of HL60 and MOLM13 cells grown for 48 hours in the absence of methionine and supplemented with dimethyl-α ketoglutarate (DiM-αKG) vitamin B12 and folic acid (n = 3). (H) ROS levels in the AML cell lines HL60, MOLM13, and K562 as determined by the total cellular ROS probes CellROX and DCF-DA and the superoxide probe MitoSOX.Buthionine sulfoximine (BSO) was used as positive control for CellROX and dichlorodihydrofluorescein diacetate (DCF-DA) and phorbol 12-myristate-13-acetate (PMA) and menadione sodium bisulfite (MSB) for MitoSOX. (I) Relative cell counts of MOLM13 cells grown in the presence or absence of methionine and N-acetylcysteine (NAC) for 24 and 48 hours (n = 4). (J) Absolute cell counts of MOLM13 cells grown with and without cysteine and in the absence or presence of 0.1 mM or 2 mM NAC for 24 hours (n = 4). (K) Graph and dotplot of ROS (DCF-DA) MFI in MOLM13 cells depleted of cysteine alone or also supplemented with 2 mM NAC (representative example from n = 3 experiments). (L) Bar graph and histogram of ROS (DCF-DA) MFI in MV4-11 cells depleted of cysteine alone or also supplemented with 2 mM NAC (representative example of n=3 experiments). Histogram representative of the 3 replicates. All experiments: error bars represent mean ± SEM. Statistical analysis by Student t test or ordinary 1-way analysis of variance (ANOVA), ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.

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