X treme transfection reagent

MCF-7 cells were purchased from ATCC. MDA-MB-231, Hs578T, T47D and 4T1 cells were available at the Luxembourg Institute of Health. These cell lines were regularly tested for mycoplasma contamination and cultured in complete growth medium following ATCC recommendations. A standard tissue culture incubator was used for a normoxic culture conditions (21% O₂; 5% CO₂). For hypoxic treatment, cells were placed in a hypoxia work station (Invivo2 400, Ruskinn) for 24 hours or 48 hours, calibrated to maintain a hypoxic atmosphere of 0.1% O₂ and 5% CO₂ by continuous flow of nitrogen. The CSRP2-depleted 4T1 and corresponding control cell lines by lentiviral transduction. CRP2 knockdown was achieved by pGIPZ lentiviral shRNAs (clone ID: V3LMM_417823 from GE Dharmacon, gene set RMM4532). A non-silencing shRNAs (RHS4346, sh-; GE Dharmacon) was used as a control. Lentivirus production was achieved by co-transfecting lentiviral pGIPZ shRNAs with packaging and envelope plasmids in HEK293T cells using Xtreme transfection reagent (Roche). 4T1 cells were infected with virus, and transduced cells were selected with 0.5 μg/ml puromycin (Sigma-Aldrich).

Elf5 was tagged at the 3′ end with V5 and incorporated into the pHUSH-ProEX vector (Genentech) [43 (link)] as descried before [16 (link)]. Elf5 expression was achieved using Doxycycline (Clontech) at 0.1 μg/ml. Luciferase/GFP [44 (link)] and pHUSH-ProEx plasmids were packed into retrovirus using PlatinumE cells (Cell Biolabs) using FuGene6 or X-Treme transfection reagent (Roche) following manufacturer instructions. PyMT cell lines were established in culture from enzymatically disaggregated PyMT tumors and double FACS-purification based on CD24 expression; and were maintained in DMEM medium containing 10%FBS, 1% L-Glutamine, 5 ug/ml Insulin, EGF 10 ng/ml, and 10 ng/ml cholera toxin. The line was considered to be established in culture after ten passages.

The macrophages were seeded into six-well plates at a density of 1 × 10⁵ cells per well and incubated for 12 h. To induce the inhibition of miR-34a, the cells were transfected with miR-34a inhibitor or negative control (NC) inhibitor (Pre-miR miRNA Precursors, Life Technologies, Karlsruhe, Germany) using X-treme transfection reagent (Roche Applied Science, Penzberg, Germany), according to the manufacturer’s protocol. The cells were harvested for further analysis 48 h after transfection, and the transfection efficiency was analyzed using qRT-PCR.

The miRNA mimic of miR-199a-3p was purchased from Invitrogen. The mirVana miRNA mimic was used to induce miR-199a-3p overexpression. Cardiomyocytes were transfected with a final concentration of 10 nmol/L for the miR-199a-3p mimic using an X-treme transfection reagent (Roche Applied Science, Penzberg, Germany) following the manufacturer's protocol.

Cardiomyocytes were seeded into 6-well plates at a density of 1 × 10⁵ cells per well and incubated for 12 h. To induce overexpression of miR-142-3p, cells were transfected with miR-142-3p mimic or negative control (NC) mimic (Pre-miR™ miRNA Precursors, Life Technologies, Karlsruhe, Germany) using X-treme transfection reagent (Roche Applied Science, Penzberg, Germany), according to the manufacturer’s protocol. The cells were harvested for further analysis 48 h after transfection, and the transfection efficiency was analyzed by qRT-PCR.