Limits...
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.

Show MeSH
Induced-mitophagy can be used to measure differential autophagy kinetics in different cell lines. (A) 3T3-SA-YFP-Parkin, Saos2 YFP-Parkin and SVEC YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin for the indicated time periods. Cell lysates were analyzed by Western blot using the MitoProfile Membrane integrity WB Antibody Cocktail, and an anti-LC3B antibody. Actin was used as a loading control. (B) Protein quantification from three independent experiments was determined by ImageJ software using the intensity of the band and expressed as mitochondria protein to actin ratio (%). ∗,∗∗Indicate significant differences between 3T3-YFP-Parkin and Saos-2 YFP-Parkin (∗p < 0.05; ∗∗p < 0.001) and $ indicates significant difference between SVEC YFP-Parkin and Saos-2 YFP-Parkin. $p < 0.05; $$p < 0.01.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4358839&req=5

f0015: Induced-mitophagy can be used to measure differential autophagy kinetics in different cell lines. (A) 3T3-SA-YFP-Parkin, Saos2 YFP-Parkin and SVEC YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin for the indicated time periods. Cell lysates were analyzed by Western blot using the MitoProfile Membrane integrity WB Antibody Cocktail, and an anti-LC3B antibody. Actin was used as a loading control. (B) Protein quantification from three independent experiments was determined by ImageJ software using the intensity of the band and expressed as mitochondria protein to actin ratio (%). ∗,∗∗Indicate significant differences between 3T3-YFP-Parkin and Saos-2 YFP-Parkin (∗p < 0.05; ∗∗p < 0.001) and $ indicates significant difference between SVEC YFP-Parkin and Saos-2 YFP-Parkin. $p < 0.05; $$p < 0.01.

Mentions: Since autophagy is the only cellular mechanism for mitochondrial removal, we were interested to know if enhanced-mitophagy could be used to reveal differences in autophagic flux between cells. Saos-2 osteosarcoma cells and SVEC endothelial cells were therefore infected with YFP-Parkin to enable mass elimination of mitochondria by autophagy and the depletion of mitochondrial proteins was measured in these and YFP-Parkin-expressing 3T3-SA cells following treatment with antimycin A and oligomycin. This revealed that not only were there significant differences in the rate of loss of mitochondrial proteins between different cell lines, but there were also differences in the rate of depletion of different mitochondrial proteins in each individual cell line Fig. 3A and B). Notably, cytochrome c and Porin (also known as VDAC1) are depleted very rapidly in SVEC and 3T3-SA after treatment with antimycin A and oligomycin (Fig. 3A and B). As a result, we did not consider them useful in determining differential rates of autophagy in these cells over this time frame.


Using enhanced-mitophagy to measure autophagic flux.

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

Induced-mitophagy can be used to measure differential autophagy kinetics in different cell lines. (A) 3T3-SA-YFP-Parkin, Saos2 YFP-Parkin and SVEC YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin for the indicated time periods. Cell lysates were analyzed by Western blot using the MitoProfile Membrane integrity WB Antibody Cocktail, and an anti-LC3B antibody. Actin was used as a loading control. (B) Protein quantification from three independent experiments was determined by ImageJ software using the intensity of the band and expressed as mitochondria protein to actin ratio (%). ∗,∗∗Indicate significant differences between 3T3-YFP-Parkin and Saos-2 YFP-Parkin (∗p < 0.05; ∗∗p < 0.001) and $ indicates significant difference between SVEC YFP-Parkin and Saos-2 YFP-Parkin. $p < 0.05; $$p < 0.01.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358839&req=5

f0015: Induced-mitophagy can be used to measure differential autophagy kinetics in different cell lines. (A) 3T3-SA-YFP-Parkin, Saos2 YFP-Parkin and SVEC YFP-Parkin cells were treated with 1 μM antimycin A + 1 μM oligomycin for the indicated time periods. Cell lysates were analyzed by Western blot using the MitoProfile Membrane integrity WB Antibody Cocktail, and an anti-LC3B antibody. Actin was used as a loading control. (B) Protein quantification from three independent experiments was determined by ImageJ software using the intensity of the band and expressed as mitochondria protein to actin ratio (%). ∗,∗∗Indicate significant differences between 3T3-YFP-Parkin and Saos-2 YFP-Parkin (∗p < 0.05; ∗∗p < 0.001) and $ indicates significant difference between SVEC YFP-Parkin and Saos-2 YFP-Parkin. $p < 0.05; $$p < 0.01.
Mentions: Since autophagy is the only cellular mechanism for mitochondrial removal, we were interested to know if enhanced-mitophagy could be used to reveal differences in autophagic flux between cells. Saos-2 osteosarcoma cells and SVEC endothelial cells were therefore infected with YFP-Parkin to enable mass elimination of mitochondria by autophagy and the depletion of mitochondrial proteins was measured in these and YFP-Parkin-expressing 3T3-SA cells following treatment with antimycin A and oligomycin. This revealed that not only were there significant differences in the rate of loss of mitochondrial proteins between different cell lines, but there were also differences in the rate of depletion of different mitochondrial proteins in each individual cell line Fig. 3A and B). Notably, cytochrome c and Porin (also known as VDAC1) are depleted very rapidly in SVEC and 3T3-SA after treatment with antimycin A and oligomycin (Fig. 3A and B). As a result, we did not consider them useful in determining differential rates of autophagy in these cells over this time frame.

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