a, Methionine deprivation increases MAT2A protein and mRNA levels. Left panel, immunoblot analysis of MAT2A and MAT1A protein expression in TNBC mammospheres grown in control or methionine-free mammosphere media for 4 days. Right panel, real-time PCR analysis of MAT2A mRNA levels in TNBC mammospheres grown in control or methionine-free mammosphere media for 72 hours. MAT2A mRNA levels were normalized to expression in TNBC cells cultured in control media. b, Immunoblot analysis of MAT2A expression in TNBC cells transfected with a scrambled control siRNA or two different siRNAs targeting MAT2A (MAT2A-1 and MAT2A-2) 48 hours after transfection. c, Mammosphere assay in TNBC cells transfected with a scrambled control siRNA or two different siRNAs targeting MAT2A (MAT2A-1 and MAT2A-2). The next day, cells were collected, seeded in ultra-low attachment plates in control or methionine-free mammosphere media (0% Met) and grown for 4 days. Left panel, representative images showing mammospheres at day 4. Right panel, mammospheres were scored in 10 fields from each well (mean ± SEM, n = 3). *, P < 0.05, **, P < 0.01, ***, P < 0.001 versus the indicated comparisons.