Fig. 3. Activation of Non-canonical NF-κB mediates ferroptosis escape by sorafenib.

Immunoblots with indicated antibodies, using cytoplasmic and nuclear lysates from WT and DDX5^KD HepAD38 cells treated ±7.5 µM SOR for 2 days (A) and transfected with 50 pM each siCtrl (-) or siNIK, as indicated, in (B). Actin used as loading control for cytoplasmic lysates and SUZ12 for nuclear extracts. C WT and DDX5^KO Huh7 cells treated ±10 µM SOR for 2 days and transfected with 50 pM each siCtrl (-) or siNIK, as indicated. A representative immunoblot is shown from n = 3. Actin used as loading control for cytoplasmic lysates and Histone 3 for nuclear extracts. D NF-κB-response element (RE) luciferase vector (pNL3.2.NF-κB-RE vector, Promega) (1.0 µg per 12-well plate) co-transfected in Huh7 WT or DDX5^KO cells with 50 pM each of siCtrl, siNIK or siβ-catenin, ±SOR (10 µM) for 48 h. Data expressed as mean ± SEM from n = 3. ***p < 0.001 by unpaired t-test. E Cell viability of Huh7 WT or DDX5^KO cells transfected with indicated siRNAs (50 pM) treated with SOR (10 µM) ±10 Ferr-1 (10 µM) for 24 h. Data expressed as mean ± SEM from n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t-test. F MDA and (G) 4-HNE abundance quantified using lysates from Huh7 WT and DDX5^KO cells, transfected with indicated siRNAs (50 pM), treated with SOR (10 µM) ±10 Ferr-1 (10 µM) for 24 h. Data expressed as SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t-test.