Fig. 4.

Shikonin enhances phosphorylation of PGC-1α, facilitating ubiquitination-mediated degradation. TNBC cells were treated with CAF-CM in the presence or absence of shikonin (2 μM) for 48 h. (A) PGC-1α expression in TNBC cells under indicated treatment. Cells were pretreated with cycloheximide (6 μM) for 12 h or MG132 (10 μM) for 6 h, and treated with or without CAF-CM ± shikonin for 48 h (n = 3). (B) Shikonin shortened the half-life of PGC-1α protein in CAF-stimulated TNBC cells. Cells were treated with cycloheximide (6 μM) with or without CAF-CM ± shikonin for the indicated time period and then harvested for western blotting (n = 3). The results were presented as fold change compared with the respective treated group at 0 h. (C) Shikonin promoted polyubiquitination of PGC-1α protein in TNBC cells. Cells were treated with MG132 (10 μM) for 12 h and harvested for western blotting. (D) Identification of phosphorylation sites of PGC-1α in TNBC cells. Immunoprecipitation experiments were conducted using an anti-PGC-1α antibody, and the phosphorylation sites of PGC-1α were identified by LC-MS/MS. (E) Alignment of PGC-1α protein sequence spanning T295 in different species. (F) Shikonin increased the interaction of PGC-1α with NEDD4-1 and GSK-3β in TNBC cells. PGC-1α was immunoprecipitated from whole-cell lysates using an anti-PGC-1α antibody, and the lysates and immunoprecipitate were blotted with PGC-1α, and NEDD4-1 and GSK-3β antibodies. (G) PGC-1α expression in TNBC cells transfected with shNC or shPGC-1α followed by transfection with PGC-1α^WT and PGC-1α^A295 plasmids, respectively. (H) Mutation of PGC-1α at Thr295 reversed the inhibitory effects of shikonin on PGC-1α expression in TNBC cells. (I) Mutation of PGC-1α at Thr295 reversed the shikonin-promoted PGC-1α polyubiquitination and its interaction with NEDD4-1 and GSK-3β in TNBC cells. Data are presented as mean ± SD, **p < 0.01, ns: not significant