br The absence of contamination with mycoplasma was tested
The absence of contamination with mycoplasma was tested routi-nely by polymerase chain reaction (PCR) assay.
The analysis was performed in MCF-7 Tet-Oﬀ/ACSL4 and MCF-7 Tet-Oﬀ empty system as previously described [20,31]. Gene expression levels were evaluated as the sum of fragments per kilobase of exon model per million reads mapped (FPKM) in each exon. For more ac-curate results, data showing estimated FPKM values under 1.0 in either cell line were filtered out (cf. the lowest level of expression commonly used is 0.05 FPKM).
2.4. Quantitative reverse transcription-PCR (qRT-PCR)
qRT-PCR or real-time PCR assays were conducted as previously described with slight modifications . MCF-7 Tet-Oﬀ empty vector and MCF-7 Tet-Oﬀ/ACSL4 total RNA was extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA). Residual genomic DNA was removed by treating RNA with DNase I (Invitrogen, Carlsbad, CA, USA) and 2 μg of total RNA were reverse transcribed using random hexamers and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocol. For real-time PCR, gene specific primers were obtained from RealTimePrimers.com (Elkins Park, PA, USA) and real-time PCR was performed using Mic PCR (Bio molecular Systems, Australia). All reactions were performed in
triplicate. Amplification was initiated by a 3-min incubation at 95 °C, followed by 45 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Gene mRNA expression levels were normalized to human 18S RNA expression, performed in parallel as endogenous control. Real-time PCR data were analyzed by calculating the 2− Ct value (comparative Ct method) for each experimental sample.
2.5. Cell sensitivity assays
Chemotherapeutic drug eﬀects on cell survival and proliferation were measured respectively by the MTT assays on cell viability  and by BrdU incorporation assays on cell proliferation following the manufacturer’s instructions. Briefly, for both assays Ifenprodil hemitartrate were plated at a density of 1500 cells/well in 96-well plates with 10% FBS-supple-mented D-MEM and allowed to adhere overnight at 37 °C in a humi-dified 5% CO2 atmosphere. The medium was then changed to serum-free medium. After 24 h, the cells were switched to 10% FBS-supple-mented D-MEM and incubated for diﬀerent times according to experi-ments. Absorbance in each assay was determined using a Synergy HT multi-detection microplate reader from Biotek (Winooski, VT, USA).
Western blot was performed as previously described . Briefly, cell cultures were washed with PBS, scraped in RIPA buﬀer and cen-trifuged at 5000g for 10 min. Total protein lysis (40 µg) was separated on SDS-PAGE, electro-transferred to polyvinylidene fluoride mem-branes and then incubated with 1% fat-free powdered milk in 500 mM NaCl, 20 mM Tris-HCl (pH 7.5) and 0.5% Tween 20 for 60 min at room temperature, with gentle shaking. The membranes were then rinsed twice in 500 mM NaCl, 20 mM Tris-HCl (pH 7.5) and 0.5% Tween 20 and incubated overnight at 4 °C with the following primary antibody dilutions: 1:1000 rabbit polyclonal anti-ABCG2, 0.5 µg/ml goat poly-clonal anti-ABCC8, 1 µg/ml goat polyclonal anti-ABCC4 and 1:5000 mouse monoclonal anti-tubulin. Bound antibodies were developed by incubation with secondary antibodies 1:5000 goat anti-rabbit, 1:5000 rabbit anti-goat and 1:5000 goat anti-mouse horseradish peroxidase-conjugated and detected by chemiluminescence. The immunoblots were then quantitated using Gel Pro Analyzer.
2.7. Intracellular doxorubicin accumulation and eﬄux assay by flow cytometry
Doxorubicin fluorescence was examined to determine intracellular accumulation. MDA-MB-231 shRNA-ACSL4, MDA-MB-231 mock (in the presence or absence of 1 µM triacsin C), MCF-7 Tet-Oﬀ/ACSL4 and MCF-7 Tet-Oﬀ empty vector cells (2 × 106) were incubated in DMEM with 1 μM doxorubicin and protected from light exposure for 1 h. Cells were then washed and incubated in doxorubicin-free medium for 2 h to assess doxorubicin eﬄux. After trypsinization, cells were harvested, washed twice and resuspended in PBS containing 2% v/v fetal bovine serum. Doxorubicin autofluorescence was immediately analyzed by FACSCalibur II flow cytometry (Becton Dickinson Biosciences) using excitation with argon laser, and detected on an FL2 channel (excitation 488 nm, emission 530 nm).
2.8. Hoechst exclusion assay
Hoechst 33342 dye was used for the Hoechst exclusion assay fol-lowing the protocol of Kong et al. (2015) with minor modifications . Cells (2 × 104) were incubated in DMEM with 5 µg/ml Hoechst 33342 dye and protected from light for 10 min at 37 °C in a culture hood. Then, cells were washed and incubated in Hoechst 33342-free medium for 10 min to assess Hoechst eﬄux. Hoechst staining was monitored by epifluorescence microscopy using the 405 nm excitation channel.