Fig 2: (A) MDA-MB-231 and MRC-5 cells were co-cultured for 72 h and both cell lines were assessed via Western blot for CAF activation markers (α-SMA and FAP) and master metabolic regulators (HIF-1 α and c-Myc); (B) A schematic representation depicting HIF-1α as the master regulator of metabolism, regulating key metabolic pathways and showing its differential expression in cancer cells and fibroblasts within the co-culture system; (C) A general scheme of cellular energy metabolism is shown, highlighting key enzymes (indicated in boxes) whose expression levels were evaluated in the co-culture experiments. Co-cultures were assessed for (D) glutamine metabolic enzymes (GLUL, GDH and ASCT2); (E) TCA cycle enzymes (Aconitase, SDHA, CS, IDH2, fumarase and MPC2); (F) glycolysis and other metabolic enzymes (GLUT1, HKII, PKM2, LDHA, PDH, MCT1, and MCT4). (A,D–F) Results are represented as mean ± SD (n = 3). Significance relative to control cells: * p < 0.05, ** p < 0.01, *** p < 0.001. The uncropped original Wester blotting images can be found in File S1.

Fig 3: MDA-MB-231 and MRC-5 fibroblast co-cultures were treated with 20 nM of VD for 72 h and cancer cells assessed via Western blot for expression of (A) glutamine metabolic enzymes (GLUL, GDH, and ASCT2); (B) TCA cycle enzymes (Aconitase, SDHA, CS, IDH2, fumarase, and MPC2); (C) other metabolic enzymes (PKM2, PDH, LDHA, MCT4, and VDR) and master metabolic regulator, HIF-1α. (D) Fibroblasts were also examined for expression of metabolic enzymes (VDR, HKII, GLUT1, LDHA, MCT1, and MCT4) and master metabolic regulator, HIF-1α, following co-culture. (A–D) Results are represented as mean ± SD (n = 3). Significance relative to control cells: * p < 0.05, ** p < 0.01. The uncropped original Wester blotting images can be found in File S1.

Fig 4: MDA-MB-231 and MRC-5 fibroblasts co-cultures were treated with 20 µM of VE for 72 h and cancer cells assessed via Western blot for expression of (A) glutamine metabolic enzymes (GLUL, GDH, and ASCT2); (B) TCA cycle enzymes (Aconitase, SDHA, CS, IDH2, fumarase, and MPC2); (C) other metabolic enzymes (PKM2, PDH, LDHA, and MCT4) and master metabolic regulator, HIF-1α. (D) Fibroblasts were also examined for metabolic enzymes (GLUT1, HKII, LDHA, MCT1, and MCT4) and master metabolic regulator, HIF-1α, following co-culture. (A–D) Results are represented as mean ± SD (n = 3). Significance relative to control cells: * p < 0.05, ** p < 0.01. The uncropped original Wester blotting images can be found in File S1.