Dear all,
How would you all interpret the following results?
Please note the cells were incubated for 24 or 48h in conditioned medium in the presence of chloroquine during whole time-course of incubation.
Furthermore, in a parallel experiment, we have observed the conditioned medium of the cells overexpressing "X" increases the gene expression of several markers of autophagy, which might make hard the conclusion of these results.
Thank you very much in advance.
You are missing a positive control for autophagy activation. CQ works (see lanes 7 and 8 in both blots), but you get strange results with GFP- and X-conditioned media (normally, you would expect the CQ lanes + other triggers to be same intensity or stronger, not less intense)
Hi Lorenzo,
Thank you very much for your message. What do you think if I run a new set of an experiment using serum starvation as positive control in the presence or absence of chloroquine for my model?
I just have an important doubt concerning if I should treat the cells with chloroquine during the whole period of conditioned medium incubation (24h and 48h), or if should I treat the cells with chloroquine on the last 6 to 3 hours before finishing the experiment. I have fear 24h or 48h of chloroquine exposure for the cells might be too toxic and associated with the conditioned medium, it could be a confounder factor of the possible results. What is your opinion about it?
Best regards and thank you very much in advance,
Juliano
1. I agree with Lorenzo; you need to use a positive control as, at first glance, it seems that LC3II/LC3I ratio has not clearly increased in CQ (-) experiments, and this may rule out a role for autophagy machinery. However, if you observed an increase in ATG expression levels for the cells overexpressing "X", the possibility of non-canonical autophagy pathways can also be taken into account. Using different autophagy blockers such as BafA1, and reprobing with p62/SQSTM1 can be helpful to have a better picture of what is going on.
2. It might be also helpful if you study shorter time courses (eg., 3-12 h): i) high doses of CQ might be toxic when exposing to the cell for longer times, and ii) an increase at the autophagy level may return to the basal level after 24-48 h in your system that is conditioned medium.
The positive control is of course very important. However to calculate autophagy flux first calculate intensity bands, normalize to actin and at the end autophagy flux calculate like this: LC3-II (with autophagy inhibitor in each conditions) - LC3-II (without autophagy in the same conditions). You will get the real numbers so then you can get informations about the real increase in autophagosome formation.
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