siRNA transfected cells were cultured during 16h with 500 nM Rapamycin. I did a WB for p62 and LC3-II. The siRNA increased both p62 and LC3-II. Rapamycin increased LC3-II as expected by autophagosome accumulation, but p62 remained unchanged. I was expecting a decrease like after starvation.
Anyone can Help ?
I have the similar results several times using cancer cell lines including PANC-1. Depending on the cell line, p62 expression is not so sensitive as LC-3 conversion. That is probably because of the transcriptional regulation of p62. SiRNA-mediated treatment might increase the p62/SQSTM1 mRNA.
Thank you for your answer. I will performed a quick RT-PCR to check its expression level.
Regards
Hi Olivier,
I agree with Go J Yoshida, so in addition you can check the protein levels of phosphorylated p62 (p-p62). In that case there might be an increase in the p-p62 but no visible decrease of p62 due to quick replenishment.
As far as I know selective autophagy is regulated by S403-phosphorylation of p62. You can see its accumulation in ATG5 KO mice.
Good luck,
Yosif
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