Figure 5. Perhexiline, but not rapamycin, causes cell death in osteosarcoma and other PHGDH-high cell lines when combined with PHGDH inhibition.

(A) Nuclear red count in NOS1 cells treated with DMSO (vehicle control) or 10 μM rapamycin.

(B) Percentage of cell death at 72 h in NOS1 cells treated with DMSO (vehicle control), NCT-503, rapamycin, or a combination of NCT-503 and rapamycin.

(C) Total protein levels of RSP6 in NOS1 cells treated with NCT-inactive, perhexiline, rapamycin, or a no treatment serum control.

(D) Nuclear red count in NOS1 cells treated with DMSO (vehicle control), 5 μM perhexiline, or 10 μM perhexiline.

(E) Percentage of cell death at 72 h in NOS1 cells treated with NCT-inactive, NCT-503, 5 μM perhexiline, or a combination of NCT-503 and 5 μM perhexiline.

(F) Percentage of cell death at 72 h in NOS1 cells treated with 10 μM PKUMDL-WQ-2101, 5 μM perhexiline, or a combination of PKUMDL-WQ-2101 and 5 μM perhexiline.

(G) Plot of combination index (CI) against fractional effect for the interaction of increasing doses of NCT-503 combined with increasing doses of perhexiline in NOS1 cells. CI > 1.0, antagonistic; 1.1 < CI < 0.9, additive; CI < 0.9, synergistic.

(H and I) Percentage of cell death at 72 h in MDA-MB-468 (H) and MDA-MB-231 (I) cells treated with NCT-inactive, NCT-503, perhexiline, or a combination of NCT-503 and perhexiline.

(J) Tumor volume of U2OS xenografts under various conditions: vehicle (n = 10), NCT-503 (n = 10), perhexiline (n = 10), and NCT-503 combined with perhexiline (n = 10).

Bars represent means of values; error bars represent SEM. All assays were conducted with n = 3 replicates unless otherwise specified. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.