Autophagy detection kit

An autophagy detection kit was purchased from Enzo life sciences (Lausen, Switzerland). Cells were cultured with vehicles or specified reagents. A positive control and negative control were added as per product protocol. Cells then were washed with Dulbecco’s phosphate-buffered saline, centrifuged, and resuspended. Cyto-ID Green Detection Reagent was added. Flow cytometric analysis was done as per product protocol.

The YOM38 yeasts containing the pRS316-GFP-ATG8 plasmid were inoculated in YPD overnight. The cultured cells at OD₆₀₀ value of 0.1 were transferred to SD medium supplemented with RES at a dose of 10 µM or dendrobine at doses of 0, 0.1, 1 or 10 µM. After incubation for 22 h, the yeasts were collected and stained by DAPI with final concentration of 20 µg/mL. The dyed yeasts were washed with PBS, suspended in 30% glycerin solution, and imaged using a two-photon confocal fluorescence microscope (Olympus FV1000BX-51, Tokyo, Japan).
For K6001 and Δrim15 of K6001 yeast, two kinds of yeasts were recovered in galactose liquid medium overnight respectively. Then, the cultivated yeast with OD₆₀₀ value of 0.1 was cultured in galactose liquid medium and treated with 10 µM RES or 0 and 1 µM dendrobine for 22 h. According to the instructions of the autophagy detection kit (Enzo Life Sciences, New York, NY, USA), the yeasts were washed with PBS and dyed using the green detection reagent in the dark for 1 h. Then, the yeasts were washed thrice and stained with DAPI. The two-photon confocal fluorescence microscope (Olympus FV1000BX-51, Tokyo, Japan) was utilized to visualize autophagy flux in yeast.

The phenotype of pDC and CD1c⁺ mDC populations was determined by flow cytometry. DC purity was assessed by double staining CD11c⁺/CD1c⁺ for CD1c⁺ mDCs (above 95%) and BDCA2/CD123 for pDCs (above 95%; all Miltenyi Biotec) (25 (link)). The following primary monoclonal antibodies (mAbs) were used to determine the maturation state of the DCs: anti–CD80-APC, anti–PD-L1-APC (all BD Bioscience, San Jose, CA). Anti-BNIP-3 Antibody (#sc-56167 FITC) was purchased from Santa Cruz Biotechnology. Anti-Mfn2 (#M6444) and Anti-Drp1 (#ABT155) were purchased from Sigma-Aldrich Anti-Porin (#529536) was purchased from Calbiochem. Anti-NDUFA10 (#ab174829) was purchased from abcam. Autophagosomes were detected using Autophagy detection kit (Enzo Life Sciences # ENZ 51031-0500) according to the manufacturer's instructions. Briefly, cells were incubated with CYTO-ID Green autophagy detection dye (1:2,000) for 30 min at 37°C. Subsequently, cells were washed and analyzed by flow cytometry. Cell viability was determined using Fixable Viability Dye eFluor™ 780 (Invitrogen # 65-0865-14) according to manufacturer's instructions. Briefly, cells were incubated with Fixable Viability Dye eFluor™ 780 (1:2000) at 4°C for 20 min. Subsequently, cells were washed and analyzed by flow cytometry. Measurements were performed on FACSVerse flowcytometers (BD).