96 well plate reader

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MA, USA) analysis was performed in 96-well plates. Briefly, 1000–2000 cells/well were seeded in 100 µL of complete medium and the day after they were treated with DOX or BNS-DOX (10⁻⁵–10⁻⁹ M) for 72 h. Subsequently, cells were supplemented with 5 mg/mL thiazolyl blue tetrazolium bromide (M2128; Sigma-Aldrich) for 2 h. Thereafter, the medium was removed and cells were lysed with 100 µL of DMSO. Absorbance was recorded at 570 nm by a 96-well-plate reader (PerkinElmer, Waltham, MA, USA). Controls were normalized to 100%, and the readings from treated cells were expressed as % of viability inhibition. Eight replicates were used to determine each data point, and five different experiments were performed.

All chemical reagents (elesclomol (T6170), Q-VD-Oph (T0282), ferrostatin-1 (T6500), necrostatin-1 (T1847), and 3-methyladenine (T1879)) were purchased from Topscience (Shanghai, China).
A total of 5000 cells were plated into a 96-well plate and incubated for 24 h. Subsequently, appropriate inhibitors were added for 12 h. The cells were then exposed to Elesclomol and Cucl2 for 24 h. In addition, 100 μL of fresh medium containing 10% cell counting Kit-8 (CCK8) solution (MA0218, Meilunbio, Dalian, China) was added. The number of cells was determined by measuring the absorbance at 450 nm using a 96-well plate reader (PerkinElmer, Hopkinton, MA, USA).

Tom70 sequence was PCR amplified and subcloned into pACT vector of CheckMate™ mammalian two‐hybrid system (Promega, Madison, WI, USA) using SalI/NotI restriction sites, and Hsp90 and Tom20 DNA sequences were PCR amplified and subcloned into pBIND vector using SalI/NotI restriction sites. Tom70‐pACT/Hsp90‐pBIND and Tom70‐pACT/Tom20‐pBIND were co‐transfected with pLuc plasmid in a 1 : 1 : 1 molar ratio into HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Triplicate samples in 96‐well cell culture plates Corning (Tewskbury, MA, USA) were used in each experiment. Hsp90, R9–Hsp90, Tom20 and R9–Tom20 peptides at concentration 20 μm were added 4 h after transfection. HEK293 cells were cultivated for 48 h, washed twice with PBS and lysed with PBS containing 1% Triton X‐100. Luciferase expression was detected using a luciferase assay kit from BioThema (Huddinge, Sweden) on a 96‐well plate reader from Perkin Elmer (Upplands Väsby, Stockholm, Sweden).

WST assay was performed according to the manufacturer's instructions to determine the EC₅₀ for KML001 (Sigma). Briefly, 5000 cells per well were seeded in a 96-well plate format and cultured overnight. The cells were treated with increasing concentrations of KML001 for 24 h after which WST reagent (Roche) was added. Killing concentration was determined by measuring the absorbance at 450 nm using a 96-well plate reader (Perkin Elmer, Woodbridge, ON) 4 h after incubation with the WST reagent.

Adenosine deaminase (ADA) was measured by a commercially available spectrophotometric assay (Adenosine Deaminase assay kit, Diazyme Laboratories, Poway, CA, USA). Haptoglobin (Hp) was measured by an in-house assay based on AlphaLISA method. Calprotectin was determined by the BÜHLMANN fCal Turbo^® assay (BÜHLMANN, Laboratories AG, Schönenbuch, Switzerland). CK was measured using a commercial kit from Beckman (Beckman Coulter Inc., Fullerton, CA, USA).
The analyses were performed in an Olympus AU600 biochemistry autoanalyzer (Olympus AU600, Olympus Diagnostica GmbH, Ennis, Ireland), except cortisol, Hp and oxytocin, which were measured with the Immulyte system and/or a 96-well plate reader (PerkinElmer, Inc., Waltham, MA, USA).

PAER2 cells were stably transfected with the indicated BECN1-luciferase constructs (primer sequences are listed in Table 1) and selected for 3 weeks with 500 μg of hygromycin B (Invitrogen). Mass cultures were collected and transiently transfected with increasing concentrations of Peg3 in 24-well plates. Luciferase was detected using a Renilla Luciferase Assay kit (Biotium) and measured using a plate luminometer (PerkinElmer Life Sciences). Data were normalized to total cell protein.
For cell proliferation assays, CellTiter Aqueous One Solution Cell Proliferation Assay was used (Promega). PAER2^pcDNA and PAER2^PEG3 cells were seeded on 96-well microplates at a density of 5,000 cells/well in 100 μl of media. One Solution Reagent was added to the wells to be measured and incubated at 37 °C for 3 h each day for 4 days. Absorbance at 490 nm was recorded using a 96-well plate reader (PerkinElmer Life Sciences).