Fig. 5.

Mutation of PGC-1α at Thr295 reverses the inhibitory effects of shikonin on CAF-stimulated metastasis and mitochondrial biogenesis in TNBC cells. (A) Detection of mitochondria using MitoTracker Green FM staining and flow cytometry (n = 3). (B) Evaluation of mtDNA levels relative to ND1 and ND5, compared with SLCO2B1 and SERPINA1 as controls (n = 4). (C) Calculation of ATP content in cellular extracts using an ATP detection kit. Intracellular ATP levels were normalized to the protein concentration (n = 5). (D) Cell migration of MDA-MB-231 cells co-cultivated with CAFs at a ratio of 2:1 with or without shikonin (2 μM) treatment for 48 h. Cells were stained with CellTracker™ Green (green fluorescence). Images were captured at 0 and 48 h following wounding (magnification, ×50; scale bars, 200 μm, n = 3). (E) Migration of MDA-MB-231 cells detected using wound healing assays with CAF-CM in the presence or absence of shikonin (2 μM). Images were captured at 0 and 48 h following wounding (magnification, ×50; scale bars, 200 μm, n = 3). (F) Cell invasion ability measured using Transwell assays in MDA-MB-231 cells cocultured with CAFs after pretreatment with or without shikonin (2 μM) treatment for 6 h. (magnification, ×100; scale bars, 200 μm, n = 3). (G) Cell invasion of MDA-MB-231 cells measured using Transwell assays after CAF-CM stimulation with or without shikonin (2 μM) for 48 h (magnification, ×100; scale bars, 200 μm, n = 3). (H) 3D-culture of MDA-MB-231 cells treated with CAF-CM in the presence or absence of shikonin (2 μM) for 3 d (magnification, ×50; scale bars, 200 μm). Data are presented as mean ± SD. ^**p < 0.01, ns: not significant