Fig. 5: DDX5 downregulation enhances NRF2 protein stability and ferroptosis escape by sorafenib.

A Immunoblots of NRF2 using WT and DDX5^KD HepAD38 cells treated with DMSO or SOR (7.5 μM) for 48 h, followed by treatment with MG132 (10 μM for 4 h), extensive washing, and addition of cyclohexamide (CHX, 200 µM), as indicated. Nuclear extracts were isolated following the time course of CHX treatment and immunoblotted with indicated antibodies. A representative immunoblot from three independent experiments. Right panel Quantification of NRF2 t1/2 by ImageJ software. Error bars represent SD from n = 3. *p < 0.05, ***p < 0.001 by unpaired t-test. B Immunoblots of NRF2 in sip62/SQSTM-transfected cells. Lysates prepared from WT and DDX5^KD HepAD38 cells transfected with 50 pM siRNAs (siCtrl or sip62/SQSTM1) for 48 h, followed by addition of MG132 (10 μM for 4 h), extensive washing, and addition of CHX (200 µM) for the indicated intervals. Nuclear lysates subjected to immunoblot analysis. A representative immunoblot is shown from n = 3. Right panel Quantification of NRF2 t1/2 by ImageJ software. Error bars represent SD from n = 3. *p < 0.05, **p < 0.01 by unpaired t-test. C Immunoblots with indicated antibodies using lysates from WT and DDX5^KO Huh7 cells treated ±SOR for 24 h. A representative experiment is shown from n = 3. D Cell viability of WT and DDX5^KO Huh7 cells transfected with indicated siRNAs (50 pM), treated with SOR (10 µM) ± Ferr-1 (10 µM) as indicated, for 24 h. Data expressed as mean ± SEM from n = 3. **p < 0.01, p*** < 0.001 by unpaired t-test. E MDA and (F) 4-HNE abundance quantified using lysates from Huh7 WT and DDX5^KO cells, transfected with indicated siRNAs (50 pM), ±SOR (10 µM) for 24 h. Data expressed as SD, n = 3. **p < 0.01, ***p < 0.001 by unpaired t-test.