Fig. 4

The uptake, subcellular targeting and promoting effect of Hst1 on cell metabolic activity of Hst1 were regulated by specific endocytic pathways. To analyze the potential mechanism on uptake pathways of Hst1, HO1N1 cells were pre-incubated with five types of endocytic inhibitors including cytochalasin D (CYD, inhibitor of phagocytosis), chlorpromazine (CPZ, inhibitor of clathrin-mediated endocytosis), amiloride (AMR, inhibitor of macropinocytosis mediated endocytosis), genistein (GEN, inhibitor of caveolae-mediated endocytosis) and methyl-β-cyclodextrin (MβCD, inhibitor of lipid raft mediated endocytosis). Before imaging, nuclear DNA was stained with NucBlue™ live cell reagent. For the detection of mitochondria and ER, cells were stained with MitoTracker® Green FM and ER-Tracker Blue-White DPX. a Typical images of F-Hst1 distribution within HO1N1 cells after pre-treatment with endocytosis inhibitors: cytochalasin D, genistein and chlorpromazine, methyl-β-cyclodextrin, amiloride. Bar = 2 μm. F-Hst1 (in red); nuclei (in blue); mitochondrial (in green). b Relative intracellular fluorescence intensity of F-Hst1 was quantitated using Fiji software. c Mander’s overlap coefficients of F-Hst1 with mitochondria in the presence of endocytosis inhibitors: cytochalasin D, genistein and chlorpromazine, methyl-β-cyclodextrin, amiloride. d Cells were then incubated in the presence or absence of various inhibitors and Hst1 for 60 min. Untreated cells were used as control. The PrestoBlue solution was added into each well after 30 min, after which absorbance was measured at wavelengths 570 nm excitation and 600 nm emission. Graph depicting the effect of various endocytosis inhibitors on effects of Hst1 on the cellular metabolic activity of HO1N1 human epithelial cells was measured by the PrestoBlue assay. Data are presented as mean ± SD; ***P < 0.001, ****P < 0.000 1. ns not significant, CYD cytochalasin D, CPZ chlorpromazine, AMR amiloride, GEN genistein, MβCD methyl-β-cyclodextrin