METTL3/14 Knockdown decreased the ability of messages to recover from hypoxic stress after 4 h reoxygenation. (A) HEK-293T cells harvested after 72 h transfection with METTL3/14 siRNA (M3/14 KD) or negative control siRNA (neg) and 24 h of hypoxic conditions and either 30 min, 1, or 4 h of room level reoxygenation recovery (reoxy). 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 in the 1 and 4 h reoxygenation experiments and SEM of two experiments in the 30 min reoxygenation experiment. Fraction containing the 80S peak is marked. Paired Student's t-test indicates significant (P ≤ 0.05) decrease (*) or increase (#) compared to negative control siRNA. (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), and 1, 2, 4, or 8 h reoxygenation after hypoxia (representative of three experiments).
