Fig. 6
From: Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells
Homocysteine metabolism is redirected toward the transsulfuration pathway during methionine stress. a A schematic summarizing isotope tracer results. The majority of deuterium atoms labeled homocysteine and cystathionine, indicating a greater flux toward glutathione synthesis (green). Methionine and SAM abundances decreases (red) in both cell lines. SAH levels increase (blue) in MB468 cells only. The activated methyl group of SAM is highlighted with yellow. b Deuterated homocysteine (Hcy-d4(3,3,4,4), dashed square) was used to prepare Met-Hcy+ media for isotope tracer analysis. Metabolite structures with blue circles indicate the expected location of the deuterium atoms. Mass isotopomer distributions (MID) were calculated using the Fiehnlab MID Analyzer and validated by comparing spectra of enriched molecules to non-labeled spectra. MIDs for M+0 (black) non-labeled parent molecule and M+4 (blue), corresponding to a mass shift resulting from four deuterium atoms, are plotted for c homocysteine, d cystathionine, e methionine, f S-adenosylmethionine (SAM), and g S-adenosylhomocysteine (SAH) for MB468 and MB468res-R8 cells. h Adenosine levels for MB468 (blue) and MB468res-R8 (yellow) cells were measured by GCTOFMS. Error bars represent standard deviation. i SAM (yellow triangles) and SAH (blue circles) levels were measured by HILIC-UHPLC-qTOFMS in MB468 and MB468res-R8 cells. Scatter plots represent peak heights for individual replicates. The overlaid line graph (SAM, yellow, SAH, blue) indicates average peak heights. A minimum of two replicates were measured per sample. j MB468 and MB468res-R8 cells cultured in Met+ (“M”) or Met-Hcy+ (“H”) media were analyzed for methionine adenosyltransferase 2A and 2B (MAT2A, MAT2B) by immunoblot. k Glutathione pools are shown for MB468 and MB468res-R8 cells. Reduced (GSH, blue circles) and oxidized (GSSG, yellow triangles) glutathione species are plotted for each time point post Met-Hcy+ media switch; horizontal bars indicate averages for each replica set. Ratios of GSH/GSSG are listed above each time point. l Cysteine levels for MB468 (blue) and MB468res-R8 (yellow) cells were measured by GCTOFMS. Error bars represent standard deviation