Chloroquine

HeLa EPHB2 CTRL^CRISPR and MYCBP2^CRISPR cells were seed in 6-well plates at a density of 0.5 million cells per well. Next day, EPHB2 overexpression was induced using 1 µg/ml tetracycline in DMEM with 10% FBS for 16 hr, followed by 26 S proteasome inhibitor (MG132, 50 µM, Sigma, #474790) or lysosome inhibitor treatment (BafilomycinA1, 0.2 µM, Sigma, #B1793; Chloroquine, 50 µM, Tocris, #4109) for 6 hr.

The following reagents were used: Matrigel (BD Bioscience), DAPI/Antifade solution (CHEMICON International, Inc), Cell Tracker Green CMFDA (Invitrogen, Tokyo, Japan), Vybrant FAM caspase-3 and -7 assay kit (Molecular Probes), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Sigma-Aldrich), DAPI/Antifade solution (Millipore, Temecula, CA), chloroquine (Tocris Bioscience), complete protease inhibitor cocktail (Roshe Diagnostics,Germany), PMSF (Sigma-Aldrich). Molecular weight markers were from Thermo Scientific: 14.4–116.0 kDa (#26610) and 10–250 kDa (#26619).
Antibodies against human LC3A/B, MDM2, Bax and tubulin were from Abcam, p53 and Bcl-2 were from Sigma-Aldrich, and anti-RL2 was from BioSan-R (Novosibirsk, Russia). The secondary antibodies used in this study were HRP-conjugated goat anti-mouse and goat anti-rabbit (BioSan-R, Novosibirsk, Russia) or FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich).
The recombinant analogue of lactaptin RL2 was obtained from E.coli and purified as described previously [3] (link). The 98% purity of the isolated protein was confirmed by RP-HPLC chromatography on C5 reverse phase column (Discovery BIO Wide Pore C5, Sigma) in water (0.5% TFA)-acetonitil solvent system using HPLC Station (Bio-RAD Laboratories) as well as by RP-HPLC on C18 (ProntosSIL) using Milichrom A-02 station (EcoNova, Russia).

Cells were transiently transfected with 1 µg of plasmids in 6-well plates and 24 h post-starvation were incubated with 100 µg/mL of cycloheximide (Sigma #4859) for indicated time points. In some experiments, transfected cells were pre-incubated with 25 µM chloroquine (Tocris, Minneapolis, MN, USA #4109), 1 µM MG132 (Cell Signaling Technologies #2194), or 10 nM bafilomycin A1 (Tocris #1334) for 16 h before the 8-h cycloheximide treatment. Cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (EMD Millipore, Billerica, MA, USA #20-188) supplemented with cocktails of protease (Thermo Fisher Scientific #78429) and phosphatase inhibitors (EMD Millipore #524,625). Lysates were then centrifuged at 13,000 rpm for 5 min at 4 °C and supernatants were collected.

HUVECs (Lonza, Walkersville, MD, USA) were cultured with EGM-2 medium (Lonza) and maintained at 37 °C with 5% CO₂. Hypoxia was performed by incubating cells in an incubator with 1% O₂ (Thermo, Middletown, VA, USA). Human PlGF and VEGF-E were purchased from ProSpec (East Brunswick, NJ, USA). Cycloheximide and chloroquine were purchased from Tocris (Bristol, UK), bafilomycin-A (BafA) was from Sigma (St. Louis, MO, USA). HeLa cells were obtained from ATCC (Manassas, VA, USA) and cultured in 10% FBS (ThermoFisher Scientific, Waltham, MA, USA) containing DMEM (ThermoFisher Scientific).

To monitor autophagy, cells were labeled with Premo Autophagy sensor LC3B-RFP (BacMam 2.0) at a MOI of 10 (Thermo Fisher Scientific, #P36236). 48 h later, live imaging was conducted with a 40 × water objective at 512 × 512 resolution for RFP and EGFP channels. Plates were then fixed with methanol and stained with mouse anti-p62 antibody and goat anti-mouse Alexa Fluor 647. Fixed plates were imaged again with the same setting with additional 647 channel for the screen.
BafA1, chloroquine, rapamycin, Torin1, and wortmannin (Tocris Bioscience) were dissolved in DMSO at stock concentrations resulting in 0.1% DMSO final concentrations. Serial dilutions were carried out with DMSO and then with culture medium to keep the DMSO concentration constant.

TM was purchased from Sigma (St. Louis, MO, USA). Resveratrol was obtained from Calbiochem (Darmstadt, Germany). Rapamycin, chloroquine and DAPI were obtained from Tocris (Bristol, UK). Antibody against GRP78 was purchased from Bioworld (St. Louis Park, MN, USA). Antibodies against CHOP, phospho-JNK, cleaved-caspase-12 and β-actin were obtained from Santa Cruz (Santa Cruz, CA, USA). Antibodies against Beclin1, LC3, LC3II, and Sirt3 were obtained from Cell Signaling (Danvers, MA, USA).