Figure 4 from N-Myristoytransferase Inhibition Causes Mitochondrial Iron Overload and Parthanatos in TIM17A-Dependent Aggressive Lung Carcinoma

NMTi treatment induces parthanatos in (KL/K)MUT lung carcinoma cells. A, Ultrastructure of (KL/K)MUT cells treated with 1 μmol/L DDD85646 (NMTi) or vehicle control for 72 hours. Bottom, Magnification of areas in the squares. Arrowheads, mitochondria. Bars, 1 μm (top) and 0.5 μm (bottom). B, Diagram of the parthanatos components. C, Detection of PARylation (PAR) by immunoblotting in lysates from (KL/K)MUT cells treated with 1 μmol/L PCLX-001 (NMTi) for 72 hours. Actin, loading control. D, Detection of PARylation (PAR) by immunoblotting in lysates from (KL/K)MUT H460 and (KL/K)WT H1437 cells expressing Tet-inducible NMT1 shRNA treated with or without Dox. GAPDH, loading control. Note that GAPDH is identical to that in Fig. 1E because the same membrane was used to stain NMT1 and PAR. E, MIF subcellular localization in (KL/K)MUT H1792 cells treated with vehicle control of 1 μmol/L PCLX-001 (NMTi) for 96 hours and processed for immunofluorescence using a MIF antibody. Representative images are shown. Bar, 5 μm in left column, 15 μm in right column. F, AIF1 immunoblotting in cytoplasmic and nuclear lysates of (KL/K)MUT H460 treated with 1 μmol/L PCLX-001 (NMTi) o vehicle for 72 hours. Tubulin and lamin staining were used to verify fraction purity. (*) non-specific band. G, AIF immunofluorescence in tumor sections from (KL/K)MUT H460 xenografts from animals treated with 25 mg/kg PCLX-001 (NMTi) or vehicle control. Arrowheads, nuclear AIF. Bar, 10 μm. H, Cell viability (live/dead reagent) in (KL/K)MUT H460 cells treated with 1 μmol/L PCLX-001 (NMTi) or vehicle control and 20 μmol/L olaparib for 72 hours. Representative fluorescent images are shown. Bar, 30 μm. Graph, percentage of dead cells calculated from at least 5,000 cells per condition. Crossbar, group mean; error bars, SEM. ****, P < 0.0001 (Student t test).