Fig 8. Mitochondrial SIRT3 mitigates TL-induced mitochondrial dysfunction in p53-deficient cells.

(A-D) SIRT3 overexpression rescues mitochondrial respiration in TL-stimulated HCT116 p53-/- cells. P53 deficient HCT116 cells were transfected with vector, myc-SIRT3 or myc-SIRT3 (H243Y). 24 hours of post-transfection, cells were stimulated with TL (50 nM) for 6 hours and were subjected for analyzing oxygen consumption rate (OCR) using the Seahorse Bioscience Extra Cellular Flux analyzer. Data were normalized with protein content and presented as basal (A), non-ATP-linked (B), maximal (C) and non-mitochondrial respiration (D) (mean±SEM; *p<0.05; n = 3). (E-G) SIRT3, but not deacetylase deficient mutant of SIRT3, abrogates TL-induced alteration in ROS production, membrane potential and ATP level. HCT116 p53-/- cells were transiently transfected with indicated plasmids. After 24 hours of transfection, cells were stimulated with TL (50 nM) for additional 6 hours. Mitochondrial ROS (mtROS) (E), ATP level (F) and mitochondrial membrane potential (G) were measured. Relative values were presented as fold change over control ((mean±SD; **p<0.001; n = 3). (H) Cells transfected with Myc-SIRT3 or Myc-SIRT3 (H243Y) were stimulated with 50 nM of TL and cell viability was assesses using CyQuont cell proliferation assay kit. The data was presented as % viable cells over vector transfected DMSO stimulated cells. (I) Protein lysates from vector, Myc-SIRT3 or Myc-SIRT3 (H243Y) transfected HCT116 p53-/- cells were immunoblotted with Myc antisera to assess the expression of overexpressed SIRT3 or SIRT3 (H243Y) mutant.