Fig. 2

The uptake, subcellular targeting, and promoting effect of Hst1 on cell metabolic activity are regulated by GPCR and ERK signaling. Experiments to assess Hst1 function dependence on GPCR, ERK or p38 MAPK signaling were performed by pre-incubating HO1N1 cells with specific inhibitors. Then cells were incubated with or without 2 μmol·L⁻¹ F-Hst1 for 60 min and stained with NucBlue™ live cell reagent. a Typical CLSM images of subcellular localization of F-Hst1 in HO1N1 cells under the treatment with 100 ng·mL⁻¹,PTx, 10 μmol·L⁻¹ U0126, or 10 μmol·L⁻¹ SB (the inhibitors of GPCR, ERK1/2 activity, and p38MAPK, respectively). Bar = 2 μm. F-Hst1 (in red); nuclei (in blue); mitochondrial (in green). b Mean 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 PTx or U0126. d Cells were then incubated in the presence or absence of various inhibitors and 10 μmol·L⁻¹ Hst1 for 60 min. Untreated cells were used as control. The PrestoBlue solution (10 μL) 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 100 ng·mL⁻¹, 10 μmol·L⁻¹ U0126, and 10 μmol·L⁻¹ SB on Hst1-stimulated cellular metabolic activity was measured by the PrestoBlue assay. Data are presented as mean ± SD; ****P < 0.000 1. ns not significant