Doxycycline monohydrate

MCF7 and MDA-MB-231 cells were obtained commercially from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, #D6546) supplemented with 10% Fetal Bovine Serum (HI FBS; Gibco, #10082-147), 2mM Glutamax (Gibco, #35050-061), and 1% Penicillin-Streptomycin (Sigma-Aldrich, #P0781). Cells were grown at 37° C in a 5% CO₂ humidified incubator. 4-OH-Tamoxifen and doxycycline monohydrate were purchased from Sigma-Aldrich (#D1822, #579002).

Doxycycline monohydrate, apo-Transferrin human, bovine serum albumin (BSA), sodium bicarbonate (NaHCO₃), sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescence probes NHS Ester Alexa Fluro 488, 556, and 647 were obtained from Invitrogen™. MDA-MB-231 (ATCC HTB-26) and 3T3 (ATCC^® CRL-1658™)) cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS. Rabbit anti-early endosome antigen 1 (EEA-1) monoclonal antibodies and rabbit anti-Rab7 monoclonal antibodies were supplied by Cell Signaling Technology. The goat anti-rabbit secondary antibody Alexa Fluor 647 was purchased by Invitrogen.

Sodium salicylate (CAS N 54-21-7), oligomycin from Streptomyces diastatochromogenes (CAS N 1404-19-9) and doxycycline monohydrate (CAS N 17086-28-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 17-DMAG (CAS N150270-08-9) was purchased from Tebu-bio (Barcelona, Spain). AICAR (CAS N 2627-69-2), Akti-1/2 (CAS N 612847-09-3), dorsomorphin dihydrochloride (CAS N 1219168-18-9), SC79 (CAS N 305834-79-1), and (R)-(+)-etomoxir sodium salt (CAS N 828934-41-4) were purchased from TOCRIS (Bristol, UK).
AICAR, Akti-1/2, dorsomorphin, etomoxir, and SC79 stock dilutions were prepared in DMSO at 20 mM whereas oligomycin and 17-DMAG were prepared at 2 and 1 mmol L⁻¹, respectively. The drug Sodium salicylate was prepared before use in the appropriate culture media at a starting concentration of 200 µmol L⁻¹.