CCCP induced mitophagy on SH-SY5Y and PC3 cell lines

SH-SY5Y (neuroblastoma) or PC3 (prostatic adenocarcinoma) cells were cultured in a 96-well plate at a density of 200,000 cells/well for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), a mitochondrial uncoupler. After 6h of incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#1 (1X) were dispensed into each well. Following cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total PINK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

HTRF Alpha-Tubulin Housekeeping assay (64ATUBPEG/H) was run simultaneously on 4µL of 1/5 diluted lysates as a normalization tool. The HTRF signal was recorded after an overnight incubation.

In both cell types, the CCCP compound triggered a dose-dependent increase in the PINK1 protein level. These results suggest that CCCP induces PINK1 accumulation, with the expected EC50 in the low µM range.

 

 

Valinomycin induced mitophagy on SH-SY5Y and PC3 cell lines

SH-SY5Y (neuroblastoma) or PC3 (prostatic adenocarcinoma) cells were cultured in a 96-well plate at a density of 200,000 cells /well for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of Valinomycin, a mitochondrial uncoupler. After 4h of incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#1 (1X) were dispensed into each well. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total PINK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

HTRF Alpha-Tubulin Housekeeping assay (64ATUBPEG/H) was run simultaneously on 4µL of 1/5 diluted lysates as a normalization tool. The HTRF signal was recorded after an overnight incubation.

In both cell types, the Valinomycin compound triggered a dose-dependent increase in the PINK1 protein level. These results suggest that Valinomycin induces PINK1 accumulation , with the expected EC50 in the nM range.

 

 

PINK1 Signaling Pathway

In a depolarization-induced mitophagy process, PINK1 (PTEN-induced kinase 1) accumulates on the damaged outer mitochondrial membrane (OMM) and is activated by dimerization and autophosphorylation. Once activated, PINK1 phosphorylates Parkin, a Ubiquitin ligase, at position Ser65 of its Ubiquitin-like domain, as well as ubiquitinated substrates on the OMM. Among them is TOM20, which once ubiquitinated is sent to proteasome. This PINK1/Parkin pathway activation results in an accumulation of PINK1 protein, phospho-Ubiquitin (Ser65), and a decrease in the TOM20 levels.

PINK1/Parkin-mediated mitophagy ensures the removal of toxic mitochondrial products and prevents neuronal loss.