Translation rates and protein output are not affected by loss of m6A. HEK-293T cells harvested after 72 h transfection with METTL3/14 siRNA (M3/14 KD) or negative control siRNA (neg) and 24 h of normoxic (normox) or hypoxic (hypox) conditions. (A) Polysome profiling of extracts separated by differential centrifugation through sucrose gradients. qRT-PCR analysis of the fractions shows percentage of individual mRNA in each fraction. Error bars represent SEM of three experiments. Fraction containing 80S peak is marked. (B) Western blots of 50 µg of protein lysates of normoxic negative control siRNA (NNeg), normoxic METTL3/14 knockdown (NKD), hypoxic negative control (HNeg), hypoxic METTL3/14 knockdown (HKD) (representative of three experiments).
