Fig. 7: TRIB2 exclusively regulates RSL3- and erastin-induced ferroptosis independent of GPX4 and the schematic presentation of the study.

A GPX4 in SK-Hep1 cells with or without TFRC or βTrCP knockout. B CHX chase of GPX4 with or without TRIB2 overexpression in control cells and SK-Hep1 cells with βTrCP knockout. The relative expression levels of GPX4 were normalized to that of GAPDH and the data from “0 h” were arbitrary set to 100%. C, D RSL3 and erastin inhibited TRIB2-induced stimulation of PSMB5 activity. PMSB5 activity (C) and GPX4 expression (D) were measured in SK-Hep1 cells with or without TRIB2 overexpression and treated with or without RSL3 (5 µM, 12 h) or erastin (10 µM, 12 h). E Cell proliferation was evaluated by a CCK-8-based method in control cells and SK-Hep1 cells with or without TFRC knockout or βTrCP overexpression. F Changes of iron did not influence apoptosis. Caspase 3/7 activities were measured in SK-Hep1 cells with or without knockout of TFRC, or in the presence or absence of DFO (25 µM, 12 h) before further treating with or without STS (100 nM, 6 h). G DNA damage was measured by a kit from Abcam in SK-Hep1 cells with or without TFRC knockout or DFO (25 µM, 12 h) administration before further treating with cisplatin (20 µM, 24 h). H Cell death was measured by staining with Sytox Green following flow cytometry in control cells and SK-Hep1 cells with or without TRIB2 knockout or overexpression following treating with DMSO, TSZ (1:1000, 4 h) or STS (100 nM, 24 h). I Schematic presentation of the study. Briefly, βTrCP-mediated ubiquitination and followed destabilization of TFRC is essential for TRIB2 to decline labile iron in liver cancer cells. Through such mechanism, the sensitivity of cells to ferroptosis is reduced. Data were analyzed by one-way ANOVA and expressed as mean ± SD from three independent experiments. NS non-significance; **P < 0.01; ***P < 0.001; ****P < 0.0001. Images of all the immunoblots are representative of three independent experiments.