P4333 100ml

Human colorectal carcinoma HCT-116 cell line and a human breast cancer MDA-MB-231 cell line (purchased from the European Collection of Authenticated Cell Cultures—ECACC, London, UK) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (D5796; Sigma–Aldrich Chemical Company, St. Louis, MO, USA) supplemented with 10% fatal bovine serum (F4135-500ML; Sigma–Aldrich Chemical Company, St. Louis, MO, USA) and 1% penicillin/streptomycin (P4333-100ML; Sigma–Aldrich Chemical Company, St. Louis, MO, USA). Both cell cultures grew in 75 cm² culture flasks and were maintained in a humidified atmosphere with 5% CO₂ at a physiological temperature of 37 °C. The media were changed every 2 days and cells were trypsinized when necessary (0.05% trypsin–0.53 mM EDTA). After a few passages and a confluence of about 80%, the human colorectal carcinoma cells and breast cancer cells were treated with medium containing PS nanoparticles (2.2 × 10¹⁰ PSNPs/mL). The polystyrene particles used in the experiments were carboxylate-modified 40 nm (red 8793 Thermo Fisher, Waltham, MA, USA) PS-fluospheresTM. Prior to each cell culture experiment, stock solutions of PS particle were prepared as previously described [10 (link)]. After treatment incubation of 24 h, 33 h, 43 h, 52 h, and 76 h, the cells were harvested for flow cytometry analysis or cytotoxicity assay.

Wildtype HeLa cells were seeded onto 12 mm glass coverslips in a density of 100.000 cells/24 well. The cells were kept in HeLa cell medium consisting of 10% FBS (S 0615; Biochrom), 1% Hepes (15630106; Thermo Fisher); 1% Non-Essential Amino Acids (M7145-100ML; Sigma Aldrich), 1% L-Alanyl-Glutamine (03-022-1B; Neo-Lab) and 1% Penicillin/Streptomycin (P4333-100ML; Sigma Aldrich) in DMEM. The next day, the cells were transfected with HCN4-pEGFP and with either CVB3-2C/pIRES-dsRed-Express2 or CVB3-3A/pIRES-dsRed-Express2 under the use of Lipofectamine3000 Transfection reagent (L3000001, Thermo Fisher). Pharmacological treatment was started directly after transfection. The HeLa cells were either treated with 0.1% DMSO; 2.5 μM N6-Benzyladenosine (4294-16-0; Santa Cruz); 10 μM GW5074 (1381, Tocris) or 1 μM CID 1067700 (ML282; Axonmedchem) for 8 h, 12 h or 24 h. The cells were immunostained against HCN4, Rab7 and LC3, as described before, and imaged. Colocalization analysis was performed with the JACoP plugin for ImageJ. Obtained Pearson´s coefficients (r), which describe the grade of localization, were multiplied with themselves to generate (r²) which was then used to give a percentual colocalization statement [26 (link), 29 (link)].

HL1 cells were cultured as described previously [15 (link)] at 37 °C under 5% CO2 on fibronectin-gelatin-coated slides in Claycomb medium (51800C, Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum (10270106, GIBCO, Waltham, MA, USA), 100 U/mL penicillin, 10 mg/mL streptomycin (P4333-100 ML, Sigma, St Louis, MO, USA), 2 mM L-glutamine (35050061, Thermo, Waltham, MA, USA), 0.1 mM norepinephrine (A9512, Sigma, St Louis, MO, USA), and 0.3 mM ascorbic acid (A7631, Sigma, St Louis, MO, USA). Plasmids were nucleofected into HL-1 cells in suspension by using the Amaxa Cell Line Nucleofector Kit V (VCA-1003, Lonza, Basel, Switzerland); 106 cells per condition were transfected by adding 4 ug of the vector. Next, cells were seeded into 24-well plates, and after 48 h, they were diluted by seeding 10,000 cells on a P100 and 5000 cells on a 6-well plate. When colonies started growing, they were picked and seeded on a 24-well plate. Cells were expanded and frozen in a vial (with Claycomb medium and 10% dimethyl sulfoxide (D2650-5X5ML, Sigma, St Louis, MO, USA)) and a pellet to extract gDNA.

In this study, mouse fibroblast cells, NIH-3T3 (3T3), were used. The 3T3 cells were cultured on a cell-culture-treated polystyrene dish (3020-100, AGC Techno Glass Co., Ltd., Shizuoka, Japan) in Dulbecco’s modified Eagle medium (D-MEM, high glucose, Nakalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (26140-079, Thermo Fisher Scientific K.K., Waltham, MA, USA) and 1% penicillin-streptomycin solution (P4333-100ML, Sigma–Aldrich Co. LLC., St. Louis, MO, USA). After confirming 80% confluence, cells were suspended in DPBS (Dulbecco’s phosphate-buffered saline, Nakalai Tesque) solution and detached from culturing dishes by incubating with TrypLE Express Enzyme (12604-021, Thermo Fisher Scientific K.K.) for 3 min. Then, the detached cells were centrifuged at 1 × 10³ rpm for 5 min (himac CT 4D, Hitachi, Ltd., Tokyo, Japan), and the cells were suspended in the culture medium. The cell density in the suspended solution was about 4 × 10⁵ cells/mL.

HEK-293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) with 10% foetal bovine serum (FBS, Sigma-Aldrich F2442) and penicillin-streptomycin (Sigma-Aldrich P4333-100ML). Cells were seeded in black 96-well plates and cultured for 24 hours. On the following day, cells were transfected with 25 ng of reporter and 150 ng of activator using Turbofect (Thermo Scientific R0531) and incubated in 5% CO₂ for 24 hours. On the following day, culture medium was removed from each well and replaced with 80 μl of 1x Lysis buffer (Dual-Luciferase Reporter Assay System, Promega E1910). Plates were incubated at room temperature for 10 minutes gently rocking and then each sample was pipetted 10 times before transferring the plates to the GloMax luminometer (Promega) for the reading. The luminometer was set to inject 25 μl of Luciferase Reagent for each well with a delay of 2 seconds between injection and measuring.

Cells were cultured in U-shaped, nonadherent 96-well plates (150 μL, 5.0 × 10³ per well) in StemSpan-ACF medium (#09855, Stem Cell Technologies) supplemented with 20% BIT9500 serum replacement (#9500, Stem Cell Technologies), CC100 (#02690, Stem Cell Technologies) mix of cytokines, 50 ng/mL VEGF (#AF-100-20, PeproTech), and penicillin with streptomycin (P4333-100ML, Sigma Aldrich). Compositions of other tested media are shown in Supplementary Table 1. After 48 hours, 50 μL of fresh medium was added; then, 50 μL of medium was changed every other day. On day 6 of culture, cells were stimulated with 7.5, 15, or 30 μmol/L atorvastatin (#3776, Tocris), 7.5, 15, or 30 μmol/L resveratrol (R5010-100MG, Sigma Aldrich), 125, 250, or 500 μmol/L acetylsalicylic acid (A5376-100G, Sigma Aldrich), 2.5, 5.0, or 10 μmol/L sulforaphane (S441-5MG, Sigma Aldrich), or 1.25, 2.5, or 5.0 mmol/L metformin (#317240-5MG, Sigma Aldrich). Conditioned media were collected after 48 hours. The controls, nonconditioned media, contained the same concentrations of stimulants and were kept for 48 hours in a cell culture incubator in the same conditions as stimulated cells. Wash-out conditioned media were collected from stimulated cells after washing with PBS and reseeding on 96-well plates in the complete fresh medium. DMSO, which was used to dissolve all stimulants, was added to the control wells.