This is an unusual result for p62. It's possible late stage inhibition of autophagy by the combination of your lysosomotropic compound and bafilomycin A1 with increasing concentration is redirecting p62 for degradation by the proteasome. This could be validated by inhibiting proteasomal activity with epoxomicin and looking for an increase in p62 levels.

What will happen if u are using protease inhibitors to avoid the protein turnover than using Bafilomycin?.

We had a strange story with p38 inhibitor -SB202190, which was initially published as inducer of autophagy. But our results could show that it was actually blocking autophagy and thus leading to accumulation of p62 and LC3-II. But it was too strong accumulation suggesting that the compound was not just blocking basal autophagy, but inducing a non-productive autophagic response. Similarly if ur compound is both an inducer of autophagy (initiation) and a late stage inhibitor (as u suggest its a lysosomal inh), this effect could be possible. Hope you can get some info with Rapamycin/BafA and DrugX/pepstatin combination treatments in ur cellular system. In addition p62 cleavage or proteasomal degradation (as suggested above) could be checked.

Good Luck

please first check the mRNA levels of p62 to be sur that your compound does not affect the synthesis of p62.

best

I agree that this is an unusual result, but I would take the result with a grain of salt for one reason, and that is your utilization of Baf A. I assume that your goal is to inhibit AV degradation by elevating the lysosomal pH, however in my hands, and that of others, Bafilomycin A can have some, shall we say, odd effects independent of its role as an inhibitor of the v-ATPase, moreover its effects on lysosomal pH are negligible as I've attempted direct quantification of lysosomal pH using Baf A, and it never results in an elevated lysosomal pH. I would suggest using Concanamycin A instead which binds the proton translocation pore of the a/c subunits of the v-ATPase V0 domain. It reproducibly elevates lysosomal pH to a pH above 5.5. Basically in addition to what is suggested by others, changing your lysosomal inhibitor to something with less off target effects may clarify your picture. I would, in agreement with Manoj, also make sure you couple your treatment with Rapamycin, as there are several compounds that I have come across that can effect a response at both the induction and degradation stages of autophagy. Good luck