Celltiter glo cell viability assay reagent

Cell cytotoxicity screening was conducted as described previously [33 (link)], using CellTiter-Glo Cell viability Assay Reagent (Promega). For proliferation assay, cells were seeded at 2 × 10⁵ cells per well in 24-well plates and treated with indicated inhibitors for 5 d. The live cells were counted each day.

HA (M_w=20 kDa) was purchased from Lifecore Biomedical (Minnesota, USA). EGCG was purchased from DSM Nutritional Products Ltd (Heerlen, the Netherlands). Sorafenib tosylate was obtained from AbMole BioScience (Houston, USA). Amicon Ultra-15 centrifugal filters were purchased from Merck Millipore Corporation (Darmstadt, Germany). MesenCult-ACF and MethoCult H4434 Classic media were obtained from Stemcell Technologies (Vancouver, Canada). Dextran (M_w=70 kDa) and radioimmunoprecipitation assay (RIPA) buffer was purchased from Sigma-Aldrich (Minnesota, USA). Anti-HARE monoclonal antibody (mAb, MBL International, clone #34 − 2), anti-CD44 mAb (Thermo Fisher, clone Hermes-1) and control rat IgG (Thermo Fisher) were used as received. The following primary antibodies used for Western blotting were rabbit polyclonal unless otherwise stated: phospho-S6 (Ser235/236) and S6 (GeneTex, USA); phospho-STAT5a (Tyr694), STAT5a (C-term), phospho-ERK1/2 (Thr202/Tyr204) and ERK1/2 (Abcepta, USA); β-actin (mouse monoclonal, Proteintech, USA). Secondary antibodies (anti-rabbit/anti-mouse IgG-horseradish peroxidase conjugates) and CellTiter-Glo cell viability assay reagent were purchased from Promega (Madison, USA). RNeasy Mini kit (Qiagen, Germany), QuantiTect Reverse Transcription kit (Qiagen, Germany) and Micro BCA protein assay kit (Thermo Scientific, USA) were used per the manufacturer’s protocol.

HA (M_w = 20 kDa) was purchased from Lifecore Biomedical (Chaska, MN, USA). EGCG was purchased from DSM Nutritional Products Ltd (Heerlen, The Netherlands). Daunorubicin hydrochloride (DNR) was obtained from AbMole BioScience (Houston, TX, USA). Amicon Ultra-15 centrifugal filters were purchased from Merck Millipore Corporation (Darmstadt, Germany). CellTiter-Glo cell viability assay reagent and Caspase-Glo 3/7 assay kit were purchased from Promega (Madison, WI, USA). 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) was obtained from Invitrogen (Carlsbad, CA, USA).