Doxorubicin

The inducible BCL6 shRNA vectors were generated based on a pLVX-TetOne-Puro vector (RRID: Addgene 124797) according to standard protocols. All constructs were verified by sequencing. shRNAs sequence targeting BCL6 are available in Supplementary file 2. Recombinant human IFN-α1 (z02866) was purchased from Genscript (Nanjing, China). Recombinant IFN-γ (300-02) and anti-human IFN-γ antibody (506532) were purchased from PeproTech (Rocky Hill, USA). Etoposide (HY-13629, a topoisomerase II inhibitor), doxorubicin (HY-15142, a topoisomerase II inhibitor), cisplatin (HY-17394, a DNA synthesis inhibitor), carboplatin (HY-17393, a DNA synthesis inhibitor), taxol (HY-B0015, a microtubule association inhibitor), and gemcitabine (HY-17026, a DNA synthesis inhibitor) were purchased from MedChemExpress (Monmouth Junction, USA).

The human breast cancer cells (BT-549, Hs578T, MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7, and T47D) were obtained from the American Type Culture Collection (ATCC, USA). All cells were cultured in the recommended medium (Gibco, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 1% streptomycin/ penicillin (Beyotime, Shanghai, China) at 37 °C with 5% CO₂. To establish cisplatin (DDP)-resistant MDA-MB-231 cell line (MDA-MB-231/DDP), doxorubicin-resistant BT549 cell line (BT-549/DOX) and tamoxifen-resistant MCF-7 cell line (MCF-7/TAM), MDA-MB-231, BT-549 and MCF-7 cells were exposed to repetitive and incremental concentrations of drugs over a period of 6 months. To maintain the resistance phenotype, MDA-MB-231/DDP, BT-549/DOX, and MCF-7R cells were, respectively, cultured in the presence of 2 μM cisplatin (Med-ChemExpress, NJ, USA), 2 μM doxorubicin (Med-ChemExpress, NJ, USA), and 1 μM tamoxifen (Med-ChemExpress, NJ, USA). Arachidonic acid was obtained from Sigma-Aldrich (St. Louis, MO, USA). Giripladib, an inhibitor of cytoplasm phospholipase A2, was purchased from USBiological Life Sciences (Swampscott, MA, USA).

Z‐VAD‐FMK was purchased from Selleck (Selleckchem, Houston, TX, USA). Nutlin‐3, tenovin‐1, and doxorubicin were commercially available from MedChem Express (Shanghai, China). The DAPK siRNA and p53 siRNA were purchased from GenePharma (Shanghai, China). LV‐DAPK1‐RNAi was purchased from GeneChem (Shanghai, China). The miR‐34a‐5p mimics, inhibitor, antagomiR, and negative control duplex were synthesized by Shanghai GenePharma. The antibodies and related reagents used in this study were obtained as follows: anti‐DAPK1 (3008S for immunoblot; Cell Signaling, Danvers, MA, USA), anti‐poly ADP‐ribose polymerase (PARP) (9532S; Cell Signaling), anti‐E‐cadherin (3195T; Cell Signaling), anti‐vimentin (5741T; Cell Signaling), anti‐pDAPK‐Ser308 (D4941; Sigma, St. Louis, MO, USA), anti‐GAPDH (G9545; Sigma), anti‐p53 (sc‐126; Santa Cruz, Dallas, TX, USA), Apoptosis Western Blot Cocktail (ab136812; Abcam, Cambridge, MA, USA), anti‐Ki67(ab15580; Abcam), anti‐DAPK1 [BA3712‐1 for immunohistochemistry(IHC); Boster Biological Technology, Wuhan, China], and anti‐N‐cadherin(CDH2) (ab76011; Abcam).

SPIO nanoparticles (average diameter, 50 nm; concentration, 2 mg/mL) were purchased from Xi’an Delta Biological Technology Co., Ltd. Doxorubicin was purchased from MedChemExpress (Monmouth Junction, NJ, USA). The SPIO solution was replaced with deionized distilled water and mixed with the DOX solution under sonication. SPIO particles and DOX were incubated in a bath at 37 °C for 4 h through the natural reaction, and then phosphate buffered saline (PBS) and magnetic precipitation were slowly added to collect the FeDOX complex. To separate the well-conjugated FeDOX complex and unconjugated DOX molecules, the collected FeDOX solution was centrifuged at 11,000 ×g for 3 min, and then resuspended in PBS.

NOZ cells were obtained from the Health Science Research Resources Bank (Osaka, Japan), GBC-SD, SGC-996, and EH-GB1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, and human embryonic kidney 293 T (HEK293T) cells were purchased from the American Type Culture Collection. NOZ and GBC-SD both were P53 WT genotype (P53^+/+). GBC-SD, SGC-996, EH-GB1, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco), and NOZ cells were cultured in William’s E medium (Gibco). All cell lines were supplemented with 10% fetal bovine serum (Gibco), penicillin (100 mg ml⁻¹) and streptomycin (100 mg ml⁻¹) and were incubated in a humidified chamber with 5% CO₂ at 37 °C. All cell cultures were ensured to be mycoplasma-negative cultures by monthly mycoplasma tests and were passaged with 0.25% trypsin containing 2.21 mm ethylenediaminetetraacetic acid (EDTA) in PBS when the cells reached 80~90% confluency. Gemcitabine (GEMZAR) was purchased from Eli Lilly. Cisplatin, cycloheximide, doxorubicin, and puromycin were purchased from MedChem Express.