Fig 1: PCBP2 and TRIB2 maintain cell viability via GPX4 under OS.a Spheroid formation by the Bel-7402 and SMMC-7721 cells with or without TRIB2 knocked out, in the presence or absence of overexpressed PCBP2 (n = 3); Scale bar = 30 µm. Data were analyzed by one-way ANOVA and expressed as mean ± SD. ***P < 0.001; ****P < 0.0001. b Tumor growth in athymic mice inoculated with Bel-7402 cells with or without TRIB2 knocked out in the presence or absence of overexpressed PCBP2 (n = 5/group); Scale bar = 4 mm. The data were analyzed by Student’s t-test and expressed as mean ± SD. ***P < 0.001; ****P < 0.0001. c–h Inhibition of GPX4 diminished the ability of TRIB2 and PCBP2 to protect cells against OS. Bel-7402 cells transfected with Dox-inducible shRNA targeting GPX4 (iGPX4sh1 (c, f); iGPX4sh2 (d, g)) were pretreated with or without Dox (700 ng/ml, 24 h) (c, d, f, g). Bel-7402 cells were also pretreated with or without RSL3 (5 µM) for 5 h (e, h). Then, the cells were exposed to the indicated concentration of t-BuOOH (c–e) or diquat (f–h) for another 24 h. Cell viability was determined and calculated as described in the “Materials and methods” section (n = 3). The data were analyzed by one-way ANOVA and expressed as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001. NS non-significance.
Fig 2: Schematic diagram of the mechanism by which TRIB2 and PCBP2 modulate proteasome function to reduce Ub and protect cells against OS via GPX4.Briefly, TRIB2 cooperates and stimulates PCBP2 to reduce the global K48-Ub level. PSMB5 is the target of these proteins, and its activity is directly promoted by PCBP2. The levels of PCBP2 and GPX4 can be upregulated via suppression of their ubiquitination. Moreover, GPX4 functions as one of the terminal effectors of TRIB2 and PCBP2 to protect liver cancer cells from oxidative damage.
Fig 3: PCBP2 is essential for TRIB2 to regulate PSMB5 activity.a Venn diagram showing three biologically independent MS results from the Bel-7402 cell immunoprecipitation with anti-TRIB2 antibodies. The candidates were further screened by UniProt online software and the literature to identify potential ubiquitination-related proteins. b Representative Coomassie blue stain image of the Bel-7402 cell immunoprecipitates pulled down by anti-TRIB2 or IgG antibodies (n = 3). c Ub in the control and Bel-7402 cells with MYPT1, GRP78, PARP1, or PCBP2 knocked down. d Ub in the control and Bel-7402 cells overexpressing PCBP1, PCBP2, or PCBP3 with or without TRIB2 knocked down. e Reciprocal co-IP results of purified TRIB2 and PCBP2 proteins in vitro. TRIB1, TRIB3, PCBP1, and PCBP3 were parallel examined to exclude non-specificity. f Positive correlation between PCBP2 and TRIB2 in the liver cancer specimens. TMA was performed by IHC using anti-PCBP2 and anti-TRIB2 antibodies (n = 208). The data were analyzed by Spearman’s rank correlation. g The effects of TRIB2 on Ub level were PCBP2-dependent. Ub in the control and Bel-7402 cells with or without TRIB2 knocked out or overexpressed, in the presence or absence of PCBP2 knocking out. h PCBP2 regulated Ub via PSMB5. Ub in Bel-7402 cells with PCBP2 and PSMB5 knocked out or overexpressed, as indicated. i TRIB2 regulated PSMB5 activity via PCBP2. A proteasome activity assay kit (AAT Bioquest) was used to evaluate PSMB5 activity in the Bel-7402 and SMMC-7721 cells under the same treatment as indicated in g (n = 3). Data were analyzed by one-way ANOVA and expressed as mean ± SD. ****P < 0.0001; NS non-significance. Images of all the immunoblots are representative of three independent experiments. The relative protein levels of the conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (c, d, g, h).
Fig 4: TRIB2 regulates Ub levels via the proteasome.a Ub in the control, Bel-7402 and SMMC-7721 cells with TRIB2 knocked down or overexpressed. b Schematic representation of Ub homeostasis. c CHX chase experiments of Ub in the control and Bel-7402 cells with TRIB2 knocked down. The relative protein levels of conj & poly and mono Ub were normalized to those of GAPDH, respectively, and the “0 h” point was arbitrarily set to 100%. d–f ALP did not participate in the regulation of Ub levels by TRIB2. Ub in the Bel-7402 cells with or without TRIB2 knocked down after treatment with the ALP inhibitors 3-MA (5 mM for 24 h) and CQ (20 µM, 24 h) (d), ATG5 knockout (e), or ATG7 knockdown (f). g Ub in the lysosomes isolated from the same cells as shown in a, as measured by IB. The relative protein levels of conj & poly Ub and mono Ub were normalized to those of LAMP2 as calculated by ImageJ software and indicated just below the blots. h, i TRIB2 did not regulate Ub levels through DUBs. Control and Bel-7402 cells with or without TRIB2 knocked down or overexpressed were treated with DMSO, VLX1570 (20 µM, 24 h), and EOAI (600 nM, 24 h), respectively (h), or in the presence or absence of UCH37 and USP14 knocked down (i). j, k TRIB2 regulated Ub levels via the proteasome. Ub levels at WCL (j) and in proteasome (k) were measured in the same cells shown in a and treated with DMSO or MG132 (8 µM, 12 h). The samples in k derive from the same experiment and the blots have been processed in parallel using control beads. The levels of conj & poly Ub were normalized to that of GAPDH at WCL (j), while normalized to that of PSMB3 in the isolated proteasome (k), and the data are graphed below the blots. Data were analyzed by one-way ANOVA and expressed as mean ± SD. ***P < 0.001; ****P < 0.0001. Images of all the immunoblots are representative of three independent experiments. The relative protein levels of the conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (a, d–f, h, i).
Fig 5: TRIB2 and PCBP2 reduce K48-ubiquitination of GPX4.a CHX chase experiments of GPX4 in the control and Bel-7402 cells with TRIB2 knocked out, with or without the simultaneous overexpression of PCBP2. The relative protein levels of GPX4 were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. b TRIB2 and PCBP2 regulated GPX4 ubiquitination. GPX4 or its K48-ubiquitination and total-ubiquitination level in the WCL of Bel-7402 and SMMC-7721 cells with or without TRIB2 knockout in the presence or absence of ectopic expression of PCBP2. The K48-Ub and total-Ub of GPX4 were normalized to that of GPX4 in the GPX4-IPs by ImageJ and indicated below the blots. c PCBP2 acts as the downstream of TRIB2 to prevent the association between GPX4 and proteasome in Bel-7402 and SMMC-7721 cells. The relative GPX4 levels were normalized to PSMB3 in proteasome and normalized to GAPDH at WCL. The relative GPX4 levels were also calculated as the ratio between the one in proteasome and the one at WCL. Besides, the PSMB5 activity was also parallel examined at WCL. Data were analyzed by one-way ANOVA and expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. d, e The TRIB2 DQLVPD element and PCBP2 KH3 domain are critical to maintain GPX4 expression. GPX4 was measured by IB in the Bel-7402 cells transfected with the indicated plasmids. The relative GPX4 levels were normalized to that of GAPDH and indicated below the blots. f GPX4 in control and Bel-7402 cells with or without TRIB2 or PCBP2 knocked out in the presence or absence of overexpressed GPX4. The relative GPX4 levels were normalized to that of GAPDH and indicated below the blots. g Tumor growth in athymic mice inoculated with the same cells as shown in f. The weight and volume of a tumor are presented below the representative tumor image (n = 5/group); Scale bar = 3 mm. Data were analyzed by one-way ANOVA and expressed as mean ± SD. **P < 0.01; ****P < 0.0001. Images of all the immunoblots are representative of three independent experiments.
Supplier Page from Abcam for Recombinant Human TRIB2 protein