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.