Figure 3.

The activity and specificity of Gamitrinib requires mitochondrial DNA. A and B, Relative mtDNA copy number in paired LN229 and LN229 ρ0 cells (A), and M059K and M059K ρ0 cells (B). Data were representative of 3 biological replicates. C and D Fluorescence intensity of Mitotracker Green in LN229 and LN229 ρ0 cells (C) and M059K and M059K ρ0 cells (D). Data were representative of 3 biological replicates. E, Immunoblot analysis of OXPHOS proteins in paired parental and ρ0 cells. F and G, Mitochondrial respiration, indicated as the oxygen comsumption rate (OCR) of LN229 (F) and M059K (G) parental and ρ0 cells, was measured by a Seahorse XF96 Analyzer. Maximal respirations were determined by the sequential addition of oligomycin, FCCP, and rotenone and antimycin A, respectively. Data were representative of 4 to 5 biological replicates. H and I, The percentage of apoptosis and cell death in parental and ρ0 cells treated with or without Gamitrinib at 5 μmol/L for 3 days. Data were representative of 3 biological replicates. J and K, Relative mtDNA copy number of parental, ρ0 and ρ0 rescued cells. Data were representative of 3 biological replicates. L and M, The percentage of apoptosis and dead cells in parental, ρ0 and ρ0 rescued cells treated with or without Gamitrinib at 5 μmol/L for 3 days. Data were representative of 3 biological replicates. N and O, Mitochondrial respirations of parental, LN229 (N) and M059K (O) ρ0 and ρ0 rescued cells treated with or without 5 μmol/L Gamitrinib, were measured by a Seahorse XF96 Analyzer. Data were representative of 4 to 5 biological replicates; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.