Figure 4.

Impact of Alkbh1 overexpression on cell fate and tRNA cleavage. A: Western blotting confirmation of Alkbh1 overexpression. Lenti-encoded Alkbh1 with GFP-attached sequence resulted in a larger protein (black arrow) than the endogenous Alkbh1 (blue arrow). B: Arsenite stress (4 hours, 1 mM) resulted in more cell death in B35^ALK than in B35^Mock cells as revealed by Annexin V FACS assay (left graphs). This effect was reflected in the form of increased gross tRNA cleavage and tiRNA generation observed on SYBR gold staining (Right). C: Antimycin stress (4 hours, 200 µg/ml) resulted in equal levels of cell death in B35^Mock and B35^ALK (left: Annexin V FACS analysis) and equal levels of tRNA cleavage (Right: SYBR gold staining). (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation). Asterisk: p< 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane. FACS data were presented as fold-change unstressed cells.