Fig. 8.

WBP2 interacted with GPX4 to inhibit its lysosomal degradation. A String analysis showed that WBP2 might interact with GPX4; B CO-IP analysis confirmed the interaction between WBP2 and GPX4; C - D Western blot studies demonstrated that cisplatin-induced GPX4 degradation was attenuated by chloroquine (an inhibitor of autophagy) and NH₄Cl (an inhibitor of lysosomes), but not MG132 (an inhibitor of proteasome); E – F Western blot studies showed that the expression of GPX4 was increased in lysosomes but decreased in lysosome-free fractionation following cisplatin treatment, and those changes were modulated by the expression of WBP2; G – H Cisplatin-induced upregulation of Lamp2a and HSC70 in BUMPT cells was mitigated by WBP2 overexpression but aggravated by WBP2 knockdown, as detected by western blot studies; I - J The diagram for the truncation of WBP2 and GPX4; K - L Western blot studies showed that the truncated forms of WBP2 (Flag-tag) and GPX4 (Flag-tag) could be expressed in 293t cells, CO-IP analysis revealed that CTD of WBP2 (flag-tag) co-precipitated with GPX4, and ΔNTD of GPX4 co-precipitated with WBP2 (Myc-tag); M PPXY motifs of WBP2 (Flag-tag) were genetical deleted, and CO-IP studies showed that mutated WBP2 with ΔPPXY1 and ΔPPXY1+2 + 3 failed to bind with GPX4; N The two KEFRQ-like motifs ¹²⁴NVKFD¹²⁸ and ¹⁸⁷QVIEK¹⁹¹ were mutated into ¹²⁴AAKFD¹²⁸ and ¹⁸⁷AAIEK¹⁹¹ in GPX4 (Flag-tag), and CO-IP studies showed that WBP2 (Myc-tag) failed to co-precipitate with GPX4 (Flag-tag) when the two motifs were alternatively or simultaneously mutated; O In cisplatin-treated BUMPT cells, WBP2 overexpression disrupted the interaction between GPX4 (Flag-tag) and HSC70, as validated by CO-IP studies. DMF: dimethylformamide, CP: cisplatin, Lv-NC: empty vector lentivirus for WBP2 overexpression, Lv-WBP2: lentivirus-mediated WBP2 overexpression, Lv-shNC: empty vector lentivirus for WBP2 knockdown, Lv-shWBP2: lentivirus-mediated WBP2 knockdown, Ad-NC: empty vector adenovirus for WBP2 overexpression, Ad-WBP2: adenovirus-mediated WBP2 overexpression, Ad-shNC: empty vector adenovirus for WBP2 knockdown, Ad-shWBP2: adenovirus-mediated WBP2 knockdown. EV: empty vector, NTD: N-terminal domain, CTD: C-terminal domain, MD: middle domain, ΔCTD: a truncated protein containing MD domain and NTD domain, ΔNTD: a truncated protein containing MD domain and CTD domain, ΔPPXY1: a mutated protein with PPXY1 motif deletion, ΔPPXY2: a mutated protein with PPXY2 motif deletion, ΔPPXY3: a mutated protein with PPXY3 motif deletion, ΔPPXY1+2 + 3: a mutated protein with PPXY1, PPXY2, and PPXY3 motifs deletion.Scale bars: 100 μm.