Figure 5. REDD1 and TXNIP interact to control ROS, ATG4B and autophagy.

(a) Physical association of REDD1 with both transfected GFP-TXNIP (left) and endogenous TXNIP (right) following transfection of HA-REDD1 and IP with α-TXNIP in 293T cells. (b) The endogenous REDD1/TXNIP complex is induced by hypoxia (1% O₂, 16 h) and energy stress (2-deoxyglucose (2-DG), 20 mM, 16 h) in 293T cells. Lysates were subjected to IP with α-REDD1 antibody. (c) Increased thioredoxin (TRX) antioxidant activity in Redd1^−/− and Txnip^−/− cells, measured by reduction of insulin disulfides. Shown is the mean of three independent experiments performed in triplicate. (d) Reduced H₂O₂ in Txnip^−/− MEFs stained with CM-H2DCFDA (3 μM) followed by flow cytometry. (e) TXNIP fails to induce H₂O₂ in Redd1^−/− cells unless co-transfected with REDD1. (f) Co-transfection of REDD1 and TXNIP potently inhibits TRX activity in Redd1^−/− cells. (g) Co-transfection of REDD1 and TXNIP is sufficient to suppress ATG4B activity in Redd1^−/− cells, assessed as in Fig. 4. (h) Impaired autophagy under basal and hypoxic (1% O₂, 4 h) conditions in Txnip^−/− MEFs, shown in the absence or presence of CQ (30 μM, 4 h). (i) Reduced MMP in Txnip^−/− cells, assessed by staining with TMRE (100 nM) followed by flow cytometry. (j) Expression of REDD1 or TXNIP is sufficient to induce autophagy in transfected 293T cells. ***P<0.001, **P<0.01, by paired t-test (c–f, i) or repeated measures ANOVA (g).