Doxorubicin

Human mammary epithelial cells (24⁺HMLE, MSP, 24⁺ Ras, MSP Ras, HMLE Twist and self-isolated MSP) were cultured in 50% MEGM (Bullet Kit, Lonza), 25% DMEM high glucose and 25% F-12 (both Invitrogen™) media supplemented with 10mg/ml human hydrocortisone, 10mg/ml human insulin and 10μg/ml human EGF (all Sigma-Aldrich). Cisplatin (Neocorp), carboplatin (medac), doxorubicin (diluted in DMSO; Santa Cruz), neocarzinostatin (NCS), cycloheximide (CHX; diluted in Ethanol; both Sigma Aldrich), Trail, TNFalpha (both R&D systems), gossypol (SellectBio), Abt-737 (active biochem, both diluted in DMSO). For siRNA-mediated knock down cells were reverse transfected with 5nM Silencer Select Pre-Designed siRNA or control siRNA (all Ambion) using Lipofectamine 2000 (Invitrogen).

The HepG2 and Huh-7 parental lines (ATCC) were cultured in DMEM (1X) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Stable knock-down (KD) of macroH2A1 was achieved by lentiviral transduction 16 (link). HepG2 or Huh-7 control cells were exposed or not to conditioned media (CM) from macroH2A1 KD cells for 24 h. Chemoresistance experiments were performed by incubating cells for 72 h with 1 μM Sorafenib (SIGMA, Germany), 2 μM Doxorubicin (Santa Cruz Biotechnology, USA) or DMSO. Pentose phosphate pathway (PPP) inhibition in Huh-7 cells was achieved by incubating cells for 24-48 h with 2.5-5 μM Physcion (Sigma, Germany). Cell proliferation was measured by MTT assay 20 (link). To assess population doubling time of HepG2 and Huh‑7 cells, suspension of cells containing 300,000 cells/well were applied to 6‑well plates. Number of the cells was counted every day up to 3 days after plating.

Doxorubicin was from Santa Cruz Biotech (SC-200923A), resuspended in water at a concentration of 10 mg/mL, and then diluted in PBS to 50 μg/mL and used at concentrations indicated. Filipin III (Filipin3) was from Sigma (Cat# F4767). Dynasore was from Abcam (Cat# 120192). Methyl-B-Cyclodextrin (MBCD) was from Sigma-Aldrich (Cat# 332615). Filipin III, Dynasore, or MBCD were added to cultures at concentrations shown immediately after removing Doxorubicin. BKM-120 was purchased from Apex Bio (Cat#A3015, Batch#2) and resuspended in DMSO for a stock concentration of 2.44 mM. GDC-0941 was purchased from Apex Bio (Cat#A8210, Batch#2) and resuspended in DMSO for a stock concentration of 1.95 mM. The drug/media were changed every 2 days until timelines were completed. pHrodo red labeling kit for phagocytosis was purchased from Sartorius (Cat#4649).

To obtain the inhibitory concentration 50 (IC₅₀) of the drugs, we followed the protocol described in (30 (link)). The cells were seeded in 96 well plates (7,000 cells per well), after 24 h, they were stimulated with the drugs at different concentrations: Doxorubicin (0.25, 0.5, 0.75, 1, 1.25, and 1.5 μM) (Doxolem ^®RU, 10 mg/5 ml), Metformin (0.001, 0.1, 5, 20, 35, 50, 65 mM) (Santa Cruz Biotechnology) and Sodium Oxamate (0.01, 0.5, 5, 15, 25, 35, 45 mM) (Santa Cruz Biotechnology). Individual treatments were performed with their respective IC₅₀ previously obtained. For the triple therapy IC₅₀, we choose the IC₅₀ of each drug, to recalculate a new IC₅₀ in combination, taking as a start point the individual IC₅₀. Cells were fixed with cold trichloroacetic acid at 10% and stained with Sulforhodamine B (MP BIOMEDICALS). The optical density was measured in a microplate reader (EPOCH, Biotek) at 510 nm. The IC₅₀ was determined from a linear regression (R2 = 0.92) to obtain the gradual dose–response graphs.

To assess cell survival, cells were seeded in 6-well culture plates. After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.). Cells were then harvested by trypsinization at different time points and stained with trypan blue. Viable cells were counted using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated at least 2 times.