I am observing increased vacuole content and cellular size in PC-3 cells stably transfected with EGFP-LC3 plasmid compared to cells stably transfected with EGFP alone as assessed by bright-field microscopy.The EGFP-LC3 cells (multiple clones) also appear to be growing slower, and as a result I have been unable to perform additional diagnostic experiments (acridine orange staining, western blotting, etc.) to answer my question. Has anybody observed this? Do increased LC3 levels create more early autophagosomes that are unable to mature due to the limited concentration of additional autophagy factors that are not co-expressed?
Thanks!
I will say possible and that is why we need to have a control. Overexpression a protein may increase ER stress which can affect autophagy. But to what extent I have no idea.
my advice is to use as control de GFP-LC3 unable to be lipidated and evalute the autophagyc flux
Thanks, Ru-Jeng and Ricardo. I have forced expression of many proteins in various cell lines throughout my years without seeing this effect. I certainly cannot rule-out ER stress at this point. It is just intriguing that I am seeing this effect with LC3 - a known autophagy factor.
I think overexpression of GFP-LC3 will alter endogenous autophagic flux according to what we saw. We also noticed decresed proliferation of these stable cells. I am working on this, but right now I have get no solid results. Have you work out this question?
- Badges
- Science method