Dapgreen

According to user instructions, autophagy-detecting dyes (Dojindo, Kumamoto, Japan), such as 12.5 nM DAPGreen [35 (link)] and 125 nM DALGreen [36 (link)], were chosen to probe autophagosomes/autolysosomes and autolysosomes, respectively. After PBS washing, drug-treated cells were detected and quantified by flow cytometry.

A total of 3 × 10⁵ ESCC cells were seeded in glass-bottom cell culture dishes. Autophagy was induced by culturing in serum-free medium for 3 h or 20 h. After the supernatant was discarded, the cells were incubated with DAPGreen (Dojindo, Kumamoto, Japan) working solution for 30 min at 37 °C. After washing with serum-free medium twice, cell nuclei were stained with 1 μg/ml Hoechst 33342 (Thermo, Rockford, Illinois, USA) and examined under a confocal fluorescence microscope (Leica, Wetzlar, Germany). Images in the same panel were taken under the same excitation conditions to precisely examine autophagy under different conditions. Autophagy was also examined with the ratio of LC3B-II to LC3B-I by immunoblot analysis. The protein bands were quantified by gray scanning. Cells were also treated with 80 μM chloroquine diphosphate (CQ, MCE) for 2 h to inhibit lysosome function. The mRFP-GFP-LC3 lentiviral was obtained from GeneChem (Shanghai, China). 10⁴ ESCC cells were plated in 24-well plates, followed by incubation in 1640 with lentiviral for 24 h. LC3 puncta were detected with Zeiss LSM980 confocal microscope fitted with a × 63 oil immersion objective.

Cells were seeded at a concentration of 1 × 105/well and incubated at 37 °C for 24 h, and then the cells were exposed to abemaciclib or MPT0L145 for 24 and 48 h. The cells were harvested and fixed in cold 70% alcohol overnight at −20 °C, and then stained with propidium iodide (PI) containing 10 mg/mL RNase and analyzed by flow cytometry. For autophagy detection, cells were seeded at a concentration of 1 × 105/well and treated with DAPGreen (Dojindo Molecular Technologies, Rockville, MD, USA) at 37 °C for 30 min, followed by removal of the the supernatant and addition of the drug-containing media. After incubation for 24 h, cells were harvested and detected by fluorescence using flow cytometry. The results were processed using FlowJo.

Carbonyl cyanide m-chlorophenylhydrazone (CCCP, #C2759) was purchased from Sigma, and MitoTracker Green FM (MTG, #M7514), LTR (#L7528), and LTG (#L7526) were purchased from Invitrogen (Thermo Fisher Scientific, USA). Penicillin-streptomycin (#15140163, 10,000 units/mL), fetal bovine serum (FBS, #26140079), and Dulbecco's modified Eagle's medium (DMEM, #11965092) were all purchased from Gibco (Thermo Fisher Scientific, USA). Phosphate-buffered saline (PBS, #SH30256.01) was purchased from Hyclone (GE Healthcare Life Sciences) and the autophagosome detection dye (DAPGreen, #D676) from Dojindo Molecular Technologies, Inc (Japan). LAMP1-mGFP was obtained from Addgene (plasmid, #34831) 53 (link). The fluorescent gold nanoparticles were custom synthesized by Luna Nanotech Inc. (Toronto, Canada); the size of the gold core was 15 nm, with 5 kDa of PEG and the fluorophore Cy5 modified on the surface. The data characterizing the nanoparticles were measured by Luna Nanotech Inc. as well. The molar concentration of the nanoparticle stock solution was 1.36 × 10^-7 M, and the surface charge of nanoparticles was a negative potential of -4.19 ± 0.22 mV.