Fv1000bx 51

To check whether GTS B affected the TORC1 signaling pathway, we constructed RIM15-GFP and MSN2-GFP yeast strains with the BY4741 background, which are located downstream of TOCR1, to observe the activity or nuclear translocation of these proteins. Initially, these yeast strains stored at −30 °C were inoculated on YPD agar plates. After forming colonies on plates, the colonies of these yeasts were inoculated in YPD liquid medium and cultured in a shaker at 180 rpm and 28 °C for 10 h. Subsequently, the MSN2-GFP yeasts at 0.1 initial OD₆₀₀ in each group were treated with GTS B at 0, 1, 3 and 10 μM and rapamycin at 1 μM as positive control for 2 h. Meanwhile, the yeast of RIM15-GFP firstly was cultured for 3 days and was subsequently treated with GTS B at different concentrations for 6 h. These yeasts of each group were washed with PBS and stained with Hoeschst 33342 (final concentration, 1 μg/mL) for 7 min in the dark. Thereafter, the cells were cleaned with PBS to remove remaining Hoeschst 33342 from the background. Finally, the nuclear translocations of Msn2-GFP and Rim15-GFP were observed using a two-photon confocal fluorescence microscope (Olympus FV1000BX-51, Tokyo, Japan) or fluorescence microscope, respectively.

The YOM38 yeast strain containing the pRS316-GFP-ATG8 plasmid was primarily cultured in 20 mL YPD medium for 24 h. The yeast cells were then harvested and washed with PBS before inoculation in an synthetic defined (SD) medium with an initial OD₆₀₀ of 0.1 and treated with a positive control RES at 300 μM and EHR at 0, 3, and 10 μM. After cultivating for 22 h, the yeast cells were collected, washed, and suspended in 245 μL PBS and stained with 5 μL DAPI (1 mg/mL) in the dark for 10 min. These cells were then washed with PBS thrice and suspended in 10 μL PBS to observe the differential interference contrast and the green and blue fluorescence images under two-photon confocal fluorescence microscopy (Olympus FV1000BX-51, Tokyo, Japan). The images were acquired and analyzed by using computer software (Olympus Fluoview Ver.4.1 Viewer). The light was avoided during the experiment.

The YOM38 yeast cells containing the pR316-GFP-Atg8 plasmid yeast strain in glucose liquid medium with initial OD₆₀₀ value of 0.1 were treated with 0, 0.3, 1, and 3 µM GENI or 30 µM RES for 22 h. Cells were washed thrice with PBS and stained with 4′,6-diamidino-2-phenylindole staining solution (20 µg/µL) in the dark. After 10 min, the yeast was washed thrice with PBS and observed using a vertical two-photon confocal fluorescence microscope (Olympus FV1000BX-51, Tokyo, Japan).

The experiment was conducted as previously described [14 (link)]. The YOM38 yeast strain containing pR316-GFP-ATG8 plasmid was incubated in the dark with YPD medium and shaking at 180 rpm. After 24 h, cells were cleaned with PBS, divided into different groups with the same OD₆₀₀ value and treated with GTS B at different concentrations of 0, 1, 3 and 10 µM and RES at 300 μM as positive control. Cells were cultured for 22 h, washed with PBS and stained with DAPI (4′, 6 diamidino-2-phenylindol 20 μg/mL) for 10 min in dark, and then the cells were cleaned with PBS to remove remaining DAPI from the background. Finally, induction of autophagy of yeast was observed using a two-photon confocal fluorescence microscope (Olympus FV1000BX-51, Tokyo, Japan).