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Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

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Promotion and inhibition of autophagy can be detected in cells expressing exogenous Parkin. (A) 3T3-SA cells expressing YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin with or without addition of 100 nM rapamycin as indicated to promote autophagy. After 16 h cells were analyzed for mitochondrial protein depletion by Western blotting. The immunoblots shown are representative of what was observed in three independent experiments. (B) Saos2-YFP Parkin cells were treated with 150 μM deferoxamine mesylate (DFO) for 1 h before adding 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were then analyzed by Western blotting and are representative of the results observed on two separate occassions. (C and D) 3T3-SA YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin −/+ 10 μM chloroquine (C) or 100 nM bafilomycin (D) to inhibit autophagy. After 48 h, cell lysates were analyzed for mitochondrial protein depletion by Western blotting. Actin was used as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.
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f0020: Promotion and inhibition of autophagy can be detected in cells expressing exogenous Parkin. (A) 3T3-SA cells expressing YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin with or without addition of 100 nM rapamycin as indicated to promote autophagy. After 16 h cells were analyzed for mitochondrial protein depletion by Western blotting. The immunoblots shown are representative of what was observed in three independent experiments. (B) Saos2-YFP Parkin cells were treated with 150 μM deferoxamine mesylate (DFO) for 1 h before adding 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were then analyzed by Western blotting and are representative of the results observed on two separate occassions. (C and D) 3T3-SA YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin −/+ 10 μM chloroquine (C) or 100 nM bafilomycin (D) to inhibit autophagy. After 48 h, cell lysates were analyzed for mitochondrial protein depletion by Western blotting. Actin was used as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.

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) [22] and with deferoxamine mesylate (DFO), a hypoxia mimetic and previously described inducer of autophagy [23]. 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.


Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

Promotion and inhibition of autophagy can be detected in cells expressing exogenous Parkin. (A) 3T3-SA cells expressing YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin with or without addition of 100 nM rapamycin as indicated to promote autophagy. After 16 h cells were analyzed for mitochondrial protein depletion by Western blotting. The immunoblots shown are representative of what was observed in three independent experiments. (B) Saos2-YFP Parkin cells were treated with 150 μM deferoxamine mesylate (DFO) for 1 h before adding 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were then analyzed by Western blotting and are representative of the results observed on two separate occassions. (C and D) 3T3-SA YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin −/+ 10 μM chloroquine (C) or 100 nM bafilomycin (D) to inhibit autophagy. After 48 h, cell lysates were analyzed for mitochondrial protein depletion by Western blotting. Actin was used as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.
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Related In: Results  -  Collection

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f0020: Promotion and inhibition of autophagy can be detected in cells expressing exogenous Parkin. (A) 3T3-SA cells expressing YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin with or without addition of 100 nM rapamycin as indicated to promote autophagy. After 16 h cells were analyzed for mitochondrial protein depletion by Western blotting. The immunoblots shown are representative of what was observed in three independent experiments. (B) Saos2-YFP Parkin cells were treated with 150 μM deferoxamine mesylate (DFO) for 1 h before adding 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were then analyzed by Western blotting and are representative of the results observed on two separate occassions. (C and D) 3T3-SA YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin −/+ 10 μM chloroquine (C) or 100 nM bafilomycin (D) to inhibit autophagy. After 48 h, cell lysates were analyzed for mitochondrial protein depletion by Western blotting. Actin was used as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.
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) [22] and with deferoxamine mesylate (DFO), a hypoxia mimetic and previously described inducer of autophagy [23]. 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.

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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

Show MeSH