Ziyu II promotes autophagic flux in CRC cells
The elevated levels of LC3-II may be attributed to either autophagy
initiation or blockage of autophagic flux. Thus, we determined whether
Ziyu II induced complete autophagic flux. First, we examined the protein
levels of LC3-II under the combinatorial treatment of Ziyu II with
chloroquine (CQ), an autolysosome inhibitor. As expected, combinatorial
treatment resulted in enhanced accumulation of LC3-II (Fig.4A).
Moreover, we also detected the colocalization of LC3 (autophagosome
marker) with LAMP1 (lysosome marker) in Ziyu II-treated CRC cells. As
shown in Fig. 4B and C, CRC cells treated with Ziyu II showed obvious
colocalization of LC3 and LAMP1, suggesting that Ziyu II promotes the
fusion of the autophagosome with lysosome. To further confirm the fusion
of autophagosome with lysosome in Ziyu II-treated CRC cells, we used a
tandem monomeric mRFP-GFP tagged LC3 construct and observed increased
formation of red fluorescent autolysosomes (GFP-RFP+ signal) (Fig.
4D-F). Together, these findings suggest that Ziyu II treatment induces
complete autophagic flux in CRC cells.