Fig 1.
PGAM5 protects hearts (A-C) and brain (D-F) from ischemia-reperfusion (I/R) injury induced cell death.
I/R-induced myocardial damage was evaluated based on RPP values (A) and heart infarct size (B,C). Pgam5 WT mice n = 9, KO mice n = 10. (p <0.01 by Student t-test; Pgam5 KO mice exhibit increased infarct volumes (D, E). p<0.01 by Student t-test. Pgam5 WT mice n = 13, KO mice n = 16 after I/R induced brain damage and (F) greater neurological deficit scores. p<0.05 by Student t-test.
Fig 2.
PGAM5 deficiency exacerbates necroptosis.
(A) Phase contrast photomicrographs of WT or KO MEFs treated with DMSO (a, b) or TCZ (c, d) for 24 hr. Cell viability was evaluated by the MTT assay, and propidium iodide staining (inset c, d). (B) Cell viability results for WT (black) and KO (white) MEFs, which were treated with TCZ with or without 50 μM Nec1 or 100 μM BHA; (C) WT and Pgam5 KO MEFs were treated with DMSO or TCZ for 6 hr and then stained with the mitochondria membrane potential dependent dye DAPI (blue), anti-HMGB1 (green) and Mitotracker (red); (D) WT and Pgam5 KO MEFs were treated with TCZ for 3 hrs, and followed by purification of mitochondria. Western blots with the indicated antibodies are shown. Tubulin and VDAC are loading controls. (E) Representative TEM micrographs of necrotic WT and Pgam5 KO MEFs treated with TCZ or DMSO vehicle for 12 hrs. (F) Jurkat I2.1 (Caspase 8 deficient) cells transfected with nonspecific (NS) or PGAM5 (siPG) siRNAs were treated with 0.5 ug/ml human TNF-α for 24 hrs with or without necrostatin (Nec1), then cell death was quantified by taking the percentage outside of the life gate (indicated by the box using dot plots with PI staining on the y-axis and forward scatter on the x-axis (left panels). Quantification is shown in a bar graph (right upper panel) and the quality of the knockdown shown by Western blot (right lower panel).
Fig 3.
PGAM5 is required for the mitophagy during cell necroptosis.
(A) TEM micrographs of WT and Pgam5 KO MEFs before and after TCZ treatment. Black arrowheads indicate double-membrane structures with encapsulated mitochondria. The black arrow indicates the mitochondria. The white arrows indicate empty vacuoles. (B) Intracellular ROS level was measured by ROS dye CM-H2CFDA for 30 minutes and analyzed by flow cytometry. WT and Pgam5 KO MEFs were treated with TCZ for the indicated time and proteins from cytosolic part (C) or total cell lysates (D) were blotted for the indicated proteins. (E) Confocal photomicrographs of mtKeima-transduced WT and Pgam5 KO MEFs treated with TCZ for 12 hrs after excitation at 450 nm and at 560 nm.
Fig 4.
PGAM5 stabilizes PINK1 to protect cell from necroptosis.
(A) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (D), CCCP(C), or TCZ (T) for 3 hours were analyzed by immunoblot as above. (B) PINK1 immunoblot of mitochondrial fractions from WT and KO hearts subjected to I/R (+) or perfusion only (-). (C) Cell viability of Pink1 WT/KO MEFs was quantified by MTT assay. Black bars represent WT controls and white bars the respective KO. Cells were treated with TCZ, TCZ plus Nec1, or TCZ plus BHA for 12 hours. (D) Cell viability of WT and Pink1 KO MEF cells was transduced with scrambled (NC) or Pgam5-specific shRNA (PG) lentiviruses, treated with TCZ, and then tested for viability by the MTT assay at indicated treatments. p <0.01 by Student t-test.
Fig 5.
PGAM5 fails to stabilize PINK1 in HT-29 cells.
(A) HT-29 cells transduced with shRNA against PGAM5 were treated with TCZ to induce necroptosis. Cell viability was evaluated by the MTT assay. (B) Mitochondria in HeLa cells and HT-29 cells were stained with anti-Tomm20 and mitochondrial morphology was evaluated by confocal. (C) PGAM5 was knocked down by lentiviral shRNA in HeLa, HT-29 and SY5Y cell lines. Then PINK1 mRNA in the control (NS) and Knock-down cells (PGsh) were quantified by RT-PCR. (D) HT-29 and HeLa cells were treated with CCCP for 3 hours and PINK1 was detected by western blot. * indicated full length PINK1 and Δ indicated cleaved PINK1.(E)HT-29 control (NS) and PGAM5 knock-down cells (PGsh) were treated with CCCP for 3 hours followed by western blot for detecting indicated proteins.