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The human breast cancer cell line MDA-MB-231 is a TNBC cell line originally established from the pleural effusion of a female patient with breast adenocarcinoma. The cell line was a gift from A/Prof Andreas Möller (QIMR Berghofer Medical Research Institute). The cell line was maintained in DMEM medium supplemented with 10% FBS at 37 °C in a humid atmosphere with 5% CO₂ and 95% air, routinely confirmed negative for mycoplasma and bacteria contamination, and the cell line was authenticated using STR profiling. The human OTR expression plasmid (pCMV6-OTR, RC211797) and the empty vector (pCMV6-vector, PS100001) were purchased from Origene (Rockville, MD, USA). The human G-protein alpha 15 subunit (wild-type, GNA15), cloned into pcDNA3.1 (pcDNA3.1-GNA15), was obtained from the cDNA Resource Center (www.cdna.org, Bloomsberg, PA, USA). Plasmid transient transfections were carried out with FuGENE HD transfection reagent (Promega, Auburn, VIC, Australia). Selective siRNAs to knock down human OTR (M-005688-02-0005) and the control (non-targeted) siRNA (D-001206-13-05) were purchased from Dharmacon as SMARTpools. OTR knock-down transfection was carried out at a 25 nM final concentration of siRNA with DharmaFECT 1 transfection reagent (Dharmacon, Lafayette, CO, USA). The OTR overexpression and knock-down efficiency tested by qPCR are shown in Figure S1.

Knockdown of CD11b protein with small interfering RNA (siRNA) in immature DCs was done by transfecting immature DCs at day 2 or 3 of culture with siRNA using the transfection reagent DF4 (Dharmacon) or HiPerFect (Qiagen), respectively. The transfection reagents were removed, and the cells were used for experiments 2 days after transfection. The siRNA (Smart pool; Dharmacon) was specific for CD11b (Human ITGAM M-008008-01). A nontargeting siRNA control pool (D-001206-13-05; Dharmacon) served as a control. The transfection efficiency was determined by flow cytometry of cells transfected with siGlo RISC-Free Control siRNA (D-001600-01; Dharmacon). Silencing of CD11b expression was verified by real-time PCR and flow cytometry.

For siRNA-mediated knockdown of mouse Slc25a1, siRNAs pool targeting Slc25a1 (Dharmacon, M-054849-00-0005, target sequences: 5′-GGTTTGGGATGTTCGAGTT-3′; 5′-GAAGCAGGGCTCAAACCAA-3′; 5′-GGCCAAAGGCATTCTACAA-3′; 5′-CCGAATACGTGAAGACGCA-3′) and non-targeting siRNA pool (Dharmacon, D-001206-13-05, target sequences: 5′-TAGCGACTAAACACATCAA-3′; 5′-TAAGGCTATGAAGAGATAC-3′; 5′-ATGTATTGGCCTGTATTAG-3′; 5′-ATGAACGTGAATTGCTCAA-3′) were transfected into adipocytes with the lipofectamine RNAiMAX reagent (ThermoFisher Scientific) according the manufacturer’s instruction. Forty-eight hours later, cells were harvested and subject to western blot analysis. For the rescue experiments in this study, shRNA targeting Slc25a1 lentivirus (Sigma-Aldrich, SHCLNG-NM_153150, target sequence: 5′-CTGCGGCTTGAAGATCCTAAA-3′) and non-target shRNA lentivirus (Sigma-Aldrich, SHC016, target sequence: 5′-GCGCGATAGCGCTAATAATTT-3′) were used for knockdown of Slc25a1 in stromal vascular fraction with puromycin selection followed by adipocytes differentiation.