Using enhanced-mitophagy to measure autophagic flux.
Bottom Line: As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations.As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process.We consider therefore that this assay will prove to be a valuable resource for investigations in which autophagy is considered important and is believed to be modulated.
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.Show MeSH
Mentions: To determine if loss of CVa, Core 1 and cyclophilin D following treatment with antimycin A and oligomycin was occurring by autophagy, we sought to determine if the loss of these proteins could be affected by agents that are known to affect autophagic flux. To this end, we firstly treated cells with rapamycin which is a well characterized inhibitor of mTOR Complex 1 and a potent inducer of autophagy (Supplementary Fig. 2A and B)  and with deferoxamine mesylate (DFO), a hypoxia mimetic and previously described inducer of autophagy . Treatment with these drugs alone had little impact on the levels of mitochondrial proteins. However, in cells treated with rapamycin or DFO together with antimycin A and oligomycin there was a marked depletion of mitochondrial proteins that was greater than that observed in cells treated with antimycin A and oligomycin alone (Fig. 4A and B). The difference in loss of CVa, Core 1 and cyclophilin D caused by treatment was particularly clear, underscoring the usability of these proteins as markers of autophagic flux in these cells. A small difference was also seen for Porin in response to rapamycin, however, no change in cytochrome c levels was evident with either drug, ruling out its applicability in this assay.
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.