Fig. 5: βTrCP-mediated ubiquitination of TFRC is essential for TRIB2 to decline labile iron.

A, B TRIB2 knockout increased while TRIB2 overexpression decreased TFRC expression. TFRC was measured by qPCR and IB in Bel-7404 cells with or without TRIB2 knocked out (A) and in SK-Hep1 cells with or without TRIB2 overexpressed (B). C TRIB2 knockout prolonged while TRIB2 overexpression shortened the half-life of TFRC protein. CHX chase experiments were performed in Bel-7404 cells with or without TRIB2 knocked out and in SK-Hep1 cells with or without TRIB2 overexpressed. D TRIB2 regulated TFRC expression in a PSMB5-independent manner. TFRC was measured by IB in Bel-7404 cells with single or double knockout (DKO) of TRIB2 and PSMB5 or in SK-Hep1 cells with or without TRIB2 overexpression, in the presence or absence of PSMB5 knockout. E βTrCP but not Smurf1 and COP1 was required for TRIB2 to reduce TFRC in SK-Hep1 cells. TFRC was measured by IB before and after overexpressing TRIB2 in SK-Hep1 cells with or without knockout of Smurf1, COP1, and βTrCP. F βTrCP increased the ubiquitination of TFRC. Exogenous Ub-HA, βTrCP-FLAG, and TFRC-Myc were ectopically expressed, as indicated in HEK-293T treating with MG132 (8 µM). The conjugation of TFRC-Myc by Ub-HA was measured by IB using anti-HA antibodies in the immunoprecipitates that immunoprecipitated by anti-Myc antibodies. G TFRC could be ubiquitinated by βTrCP, as measured by an in vitro ubiquitination assay using anti-TFRC antibodies after incubation with purified proteins as indicated. H TRIB2 deletion-reduced ubiquitination of TFRC was blocked after βTrCP was simultaneously knocked out. Ubiquitination of TFRC was measured by anti-Ub antibodies in the immunoprecipitates that immunoprecipitated by the anti-TFRC antibodies in Bel-7404 cells with single or double knockout of TRIB2 and βTrCP. The immunoprecipitates that pulled down by IgG antibodies were parallel examined by anti-TFRC antibodies. I TRIB2 overexpressing-induced ubiquitination of TFRC was diminished after βTrCP was simultaneously knocked out. The measurement for the ubiquitination of TFRC was similarly performed as that in H. J, K TRIB2 altered labile iron was βTrCP-dependent. Expression of TRIB2 and βTrCP and labile iron were measured in Bel-7404 (J) and SK-Hep1 cells (K) under the treatments as indicated. Data were analyzed by Student’s t test and expressed as mean ± SD from three independent experiments. NS non-significance; ***P < 0.001. Images of all the immunoblots are representative of three independent experiments.