Attractene transfection reagent

Cells were seeded in 6-well plates or 10 cm dishes at 70% confluence prior to transfection. Transient transfections of plasmids and miRNA mimics were performed using Attractene Transfection Reagent (301005, Qiagen) following the manufacturer's instructions.

NMuMG, 4T1, and MDA-MB-231 cells were transfected with Lipofectamine 2000 (ThermoFischer) while HEK-293 cells were transfected with Attractene transfection Reagent (Qiagen). NMuMG, 4T1, and MDA-MB-231 cells stably transfected with recombinant pBICEP-CMV-2-based vectors were maintained in selective medium containing 800, 350, and 750 μg ml⁻¹ G418 (Sigma-Aldrich), respectively. NMuMG cells stably transfected with recombinant pIRES1hyg-based vectors were maintained in selective medium containing 600 μg ml⁻¹ Hygromycin B (Sigma-Aldrich). Specific mock cells were generated (for every cell line and every plasmid backbone) by transfecting the corresponding empty vector in each cell type. Mock cells were subjected to a selection procedure identical to the other transfectants. siRNAs utilized to knockdown murine EPR (5′—GAGCAAAAGAGAAUGCUUA—3′) were purchased from Thermo Fisher. Stable KHSRP knockdown in NMuMG cells was obtained using previously described silencing sequences and pSuper-Neo (Oligoengine) according to the manufacturer’s instructions^{15 (link)}. The adenoviral vectors pAdCMVnull (AdNull) and pAdKHSRP (full-length human KHSRP cDNA cloned into an Adenoviral-Type 5 backbone) were purchased from Vector Biolabs^{15 (link)}.

For high-transfection efficiency and low background expression of bba-milR1, the mammalian HEK293T cell line (ATCC) was used for luciferase reporter assay^{37 (link)}. The HEK293T cells were grown in the DMEM/HIGH GLUCOSE medium (HyClone) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS, Gibco) and 1 × antibiotic–antimycotic (Gibco) at 37 °C under 5% CO₂. The ~400 bp sequences surrounding the predicted bba-milR1 target sites in CLIPB9 and Spz4 were separately cloned into the XhoI and NotI sites of the psiCheck-2 vector (Promega). Mutagenesis PCR was performed to introduce point mutations at the bba-milR1 target sites to construct psiCheck-2-mut vectors. The HEK293T cells were transfected with 100 ng of psiCheck-2 reporters with 100 nM (final concentration) of synthetic bba-milR1 Mimic (sense strand 5′-UGACUGGCUGUUAAUUGACUGG-3′, anti-sense strand 5′-AGUCAAUUAACAGCCAGUCAUU-3′, RiboBio) or Negative Control miRNA Mimic (micrON mimic NC #24, RiboBio) using Attractene Transfection Reagent (Qiagen). Cells were collected and lysed at 48 h after transfection. The activities of firefly and Renilla luciferases were measured using the Dual-Luciferase Reporter Assay System (Promega). Each sample was performed in triplicate, and transfections were repeated three times.

The murine Raw 264.7 (ATCC), COS7 (ATCC) and HEK293T cell lines (ATCC) were cultured in DMEM containing 10% (vol/vol) heat-inactivated FBS, 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin (D10). Transfections were performed using Attractene transfection reagent according to manufacturer's instruction (QIAGEN).