Cell culture

The human pancreatic cancer cell lines Capan-1 and SW1990 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 IU/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. In serum starvation experiments, cells were washed twice with phosphate-buffered saline (PBS) and once with modified Earle’s balanced salt solution (EBSS) medium (Sigma-Aldrich, St. Louis, MO, USA) and then incubated with EBSS medium at 37 °C.

Chemicals and antibodies

The compound Her, synthesized by Dr. Zhi-guo Mang, was dissolved in dimethyl sulfoxide (DMSO) as a 50 mM stock solution and stored at −80 °C. Bafilomycin (BafA1, #HY-100558), hydroxychloroquine sulfate (HCQ, #HY-B1370) and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, #HY-D0940) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Compound C (CC, S7306) was purchased from Selleck Chemicals (Houston, TX, USA). N-acetylcysteine (NAC, #S0077) and the Annexin V-FITC apoptosis detection kit (#C1062L) were purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies against p-AMPKα (Thr172, 1:1000, #2535) and p-p70S6K (Thr389, 1:1000, #9234) were obtained from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against LC3 (1:1000, #14600-1-AP) and SQSTM1/p62 (1:1000, #18420-1-AP) were obtained from Proteintech Group (Wuhan, Hubei, China). The following antibodies were obtained from Abclonal Technology (Wuhan, China): ATG5 (:1000, #A0203), AMPKα (1:1000, #A12718), p-mTOR (Ser2448, 1:1000, #AP0094), mTOR (1:1000, #A2445), p70S6K (1:1000, #A2190), β-actin (1:5000, #AC038) and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (H + L) (1:5000, #AS014).

Cell viability assay

The Cell Counting Kit-8 (CCK-8, #C0038, Beyotime) was used to determine cell viability. Capan-1 and SW1990 cells were seeded into 96-well plates (5 × 103 cells per well) and cultured in 100 μL medium overnight at 37 °C. Cells were treated with various concentrations of Her (0 to 160 μM) for 24 h or 48 h. Next, the cells were incubated with 10 μL CCK-8 for another 2 h. Finally, the absorbance value of each well at 450 nm was detected using a microplate reader (SpectraMax ABS Plus, Molecular Devices, LLC., San Jose, CA, USA).

Western blot analysis

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (#P0013D, Beyotime), and the concentration of total protein was measured by a bicinchoninic acid protein quantitative assay kit (#P0010, Beyotime). Approximately 30 μg of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 2 h at room temperature. The membranes were incubated with specific primary antibodies overnight at 4 °C. The membranes were then incubated with the corresponding horseradish peroxidase-labeled secondary antibody at room temperature for 2 h. Protein bands were identified using the enhanced Tanon 5200 Multi chemiluminescence (ECL) system (Tanon Science and Technology, Shanghai, China). The bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and β-actin was used as an internal control.

Fluorescence microscopy

The stub-RFP-sens-GFP-LC3 lentivirus was obtained from GeneChem Co. Ltd (Shanghai, China). To construct stable cell lines expressing stub-RFP-sens-GFP-LC3, Capan-1 and SW1990 cells plated in 96-well plates were cultured overnight and then infected with lentivirus. The multiplicity of infection was 20 for Capan-1 and SW1990 cells. Infected cells were selected by 3 μg/mL puromycin (Solarbio Life Science, Beijing, China). When GFP-RFP-LC3 autophagic puncta were detected, cells were cultured on 24-well cell cover glasses, fixed with 4% paraformaldehyde for 15 min, and stained with 1 μg/mL diamidino-2-phenyl indole for 10 min. The staining process was performed at room temperature and cells were protected from light. The GFP-RFP-LC3 autophagic puncta were examined using a Leica SP5 confocal microscope (Leica Microsystems Inc., Germany) with a 63× oil immersion lens.

Transmission electron microscopy

Capan-1 and SW1990 cells were fixed with 2.5% glutaraldehyde at room temperature in the dark. The cells were gently scraped off culture dishes with a cell scraper along one direction, collected by centrifugation, resuspended in 2.5% glutaraldehyde and stored at 4 °C. The cells then underwent fixation, dehydration, infiltration embedding, polymerization, slicing and staining. The slice thickness was 60–80 nm. The sections were observed under a transmission electron microscope (TEM, HT7800, Hitachi, Japan) and images were collected for analysis.

Autophagic cell death

Cell death was measured using an Annexin V-FITC apoptosis detection kit. Briefly, Capan-1 and SW1990 cells were digested with trypsin and resuspended in Annexin V binding buffer. The cell suspension was incubated with 5 μL of Annexin V-FITC for 15 min at room temperature and away from light. Then, 10 μL of propidium iodide solution was added to the cell suspension, and the sample was mixed gently and incubated for 5 min. The percentage of cell death was determined using flow cytometry (CytoFLEX, Beckman Coulter), and the data were analyzed with Flowjo software.

Small interfering RNA

SiATG5 and siAMPKα were purchased from RiboBio Co., Ltd. (Guangdong, China). The target sequences were as follows: ATG-5: 5ʹ-GCTCTTCCTTGGAACATCA-3ʹ; and AMPKα: 5ʹ-GTGGAACCCTTCCATTTGA-3ʹ. Capan-1 and SW1990 cells were cultured in 12-well plates and transiently transfected with 150 nM siATG5 or siAMPKα using riboFECT™ CP Reagent (#C10511-05, RiboBio) in accordance with the manufacturer’s instructions.

Measurement of ROS level

Capan-1 and SW1990 cells were treated with various concentrations of Her (0, 5, 10, 20, 30, 40 μM). After 6 h or 12 h, cells were incubated with 10 µM H2DCFDA for 30 min at 37 °C in a CO2 incubator. Cells were washed three times with PBS to remove unbound probes and digested with trypsin. The intensity of dichlorodihydrofluorescein (DCF) fluorescence was detected by CytoFLEX flow cytometry (Beckman Coulter).

Statistical analysis

All experiments were repeated three times, and the data are expressed as the mean ± standard deviation (SD). All statistical graphs were generated using GraphPad Prism 7 software (La Jolla, CA, USA). Student’s t-test was used for analysis of the significant difference between the groups, and one-way analysis of variance (ANOVA) was performed for comparisons in multiple groups. P < 0.05 was considered statistically significant.