Dulbecco s modified eagle medium

Human triple negative breast adenocarcinoma cell line MDA-MB-231 was grown in Dulbecco’s modified Eagle medium (Euroclone, Italy) supplemented with 10% fetal bovine serum (GIBCO, Italy), 100 U/mL penicillin/streptomycin and 2 mM L-glutamine (Euroclone, Italy). Cells were transduced with L-LUC-IN2, a retroviral vector coding for the firefly luciferase, to allow IVIS monitoring of tumor mass, as described^{48 (link)}. Non-obese diabetic severe combined immunodeficient (NOD-SCID) mice (6–8 weeks old; Charles River, Italy) were used to evaluate pharmacokinetics and antitumor activity of the metformin formulations. Animals were handled in agreement with Italian regulations for the protection of animals used for scientific purposes and guidelines of the Ethical Committee for Animal Experimentation of the IRCCS-AOU San Martino-IST (Genova, Italy). All the following in vivo procedures were reviewed and approved by Review Board of the IRCCS San Martino-IST and by the Italian Ministry of Health (n°338, DLvo 116/92), and are compliant with EU Directive 2010/63/EU for animal experiments.
Dosage form preparation as sol or in prefilled syringeThermal and rheological characterizationDynamic light scattering assaySee supplementary information.

LX2 (a kind gift from Prof. S. Fiorucci, University of Perugia) cells were cultured at 37°C in an atmosphere of 5% CO₂ in Dulbecco's Modified Eagle Medium (Euroclone-Milan, Italy) supplemented with 10% Fetal Bovine Serum (FBS) (Euroclone), 2 mM L-Glutamine and antibiotics (penicillin/streptomycin).

Phosphate buffer saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), and Horse serum (HS) were purchased from EuroClone (Pero, Italy). Chick embryo extract was obtained from United States Biological (Salem, MA, USA). Basic fibroblast growth factor (FGFb) was purchased from PeproTech (Cranbury, NJ, USA). The primary antibodies, including their suppliers, are listed in Supplementary Table S1. The primers pairs (Supplementary Table S2) were purchased from Eurofins (Vimodrone, Italy). The cocktail of protease and phosphatase inhibitors cOmplete and PhosSTOP was obtained from Roche Applied Science (Mannheim, Germany). Horseradish-peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Fluoroshield Mounting Medium containing DAPI and fluorescent phalloidin (#ab176752) were obtained from Abcam (Cambridge, UK). DAPI, Alexa-conjugates and TO-PRO™-3 Iodide (#T3605) were purchased from Invitrogen-ThermoFisher Scientific (Waltham, MA USA). Normal goat serum was obtained from Vector Laboratories (Newark, CA, USA). All other chemicals were from Sigma-Aldrich Merck (Darmstadt, Germany).

HEK293 cells, stably expressing human TRPM8 (GenBank number NM_024080) [31 (link)], were cultured in T75 tissue culture flasks using Dulbecco’s modified Eagle medium (Euroclone, Pero, Italy), supplemented with 10% FBS (Euroclone, Pero, Italy) plus Geneticin (Sigma-Aldrich, St. Louis, MI, USA) 800 μg/mL at 37° C, in a humidified 5% CO₂ atmosphere. The cells were maintained until they reached 70~80% confluency. Prior to use, the cells were washed twice with Ca²⁺ and Mg²⁺ free PBS (Euroclone, Pero, Italy), subsequently detached using Accutase (Euroclone, Pero, Italy), and kept in suspension using CHO-SFM-II medium (Gibco, by Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were then resuspended in the external solution after a centrifugation at 1000 rpm for 2 min. Single-cells suspension, with a final concentration of 3–5 million cells/mL, was required for the automated patch clamp electrophysiology procedure.

HeLa cells, derived from human cervix cancer, and CHO, Chinese hamster ovary, cells were grown in DMEM (Dulbecco’s modified Eagle medium, Euroclone, Devon, UK) and DMEM/F-12 (Dulbecco’s modified Eagle medium/Nutrient Mixture F-12, Euroclone, Devon, UK), respectively, supplemented with 10% FBS (Gibco, Paisleg, UK), 1 mM l-glutamine (Sigma Aldrich, St Louis, MO, USA), 1mM sodium pyruvate (Biowest, Miami, FL, USA), and 100 U/ml penicillin–streptomycin (Euroclone, Devon, UK).

The HeLa cell line was grown in Dulbecco’s modified Eagle medium (Euroclone, Milano, Italy) supplemented with 10% v/v fetal calf serum (Hyclone, GE Healthcare Life Sciences, Milano, Italy). Cells were plated (18*10⁴) in a 6 well plate in antibiotic-free complete medium and transfected the following day with 50 nM DNAJC17 siRNA (ON-TARGETplus Human DNAJC17 siRNA, cat n° L-021141, Dharmacon, GE Healthcare Life Sciences, Milano, Italy) and 50 nM siRNA negative control (ON-TARGETplus Non-targeting Pool, cat n° # D-001810 Dharmacon, GE Healthcare Life Sciences, Milano, Italy) in INTERFERin transfection reagent (cat n° 409-10 Polyplus-transfection, Illkirch FRANCE) following the manufacturer’s protocol; three technical replicates were prepared for each condition. Cells were harvested 96 h after transfection and total RNA was extracted with TRIzol reagent (Thermo-Fisher, San Jose, CA, USA) for RNA-seq transcriptome analysis.