Figure 6.

REDD1/DDIT4 Regulates tRNA Retrograde Transport

(A) HeLa cells were exposed to 5 mM H₂O₂ for 2 h, and changes in RNA expression were analyzed using a pre-coated 96-well qPCR assay with probes specific for the mTOR signaling pathway. Significant up and downregulated genes (FC >1.5, p ≤ 0.05) are indicated in red and green, respectively.

(B) Inhibition of the mTOR pathway was confirmed by western blot to detect phosphorylation of the translation inhibitor 4EBP and the ribosomal protein S6 (Ser235/236).

(C) REDD1 knockout MEFs (REDD^−/−) showing reduced tRNA retrograde transport when treated with 3 mM H₂O₂ compared with wild-type cells (REDD^+/+). Arrows point to cells with a positive nuclear signal. Scale bar, 10 μm.

(D) REDD1 protein expression analysis by western Blot for REDD^+/+ and REDD^−/− cells.

(E) The N/C ratio was calculated as described in the legend for Figure 1. Unpaired two-tailed Student’s t test was used to calculate statistical significance.

(F) Cells were incubated for 4 h with the hypoxia mimetic agent desferrioxamine (DFO, 5 mM), and tRNA nuclear accumulation was detected by tFISH and quantified by ImageJ. The graphs show data from one representative experiment of three.

(G) Stabilization of HIF-1 was detected by western blot in parallel experiments, and actin was used as a loading control.

See also Table S6.