Opti mem 1

For transient transfections, U87 cells (3 × 10⁴ cells/mL) were plated in 6-well plates or Petri dishes. After 24 h, a mixture of 100 nM (final concentration) of miRNA oligonucleotide-inhibitors (negative control and miR-143) (Life Technologies, Grand Island, NY, USA), lipofectamine RNAiMAX (Life Technologies) (1:1 ratio, v/v), and Opti-MEM I (Life Technologies) was added to the cells for 6−8 h. Then, the media was changed to regular DMEM with 10% FBS. For stable transfections, T98G cells were plated in a 6-well plate (3 × 10⁴ cells/mL). After 24 h, a mixture of 3 µg of miR-143 (pCMV-MIR143) or Empty (pCMV-EV) OriGene vectors (OriGene Technologies, Inc. Rockville, MD), lipofectamine RNAiMAX (Life Technologies) (1:1 ratio, v/v), and Opti-MEM I (Life Technologies) was added to the cells for 6−8 h. These vectors contain a Neomycin resistance cassette, which was used for mammalian cell clone selection and maintenance. Independent clones were picked and grown individually. RNA was isolated using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA), following manufacturer’s instructions. To monitor miR-143 expression in empty vector and miR-143-overexpressing clones we used Taqman based qPCR (Applied Biosystems, Life Technologies, NY, USA). Threshold cycles (Ct) were used to calculate the relative miR-143 expression using U48 as an internal control [14 (link),58 ].

To determine the growth kinetics, we inoculated the MDCK and A549 cells with rgH6N6-222V/228S, rgH6N6 × pdm09-222V/228S, rgH6N6-222A/228G, rgH6N6 × pdm09-222A/228G, rgH3N2 × pdm09 (Table 1), wild-type A/California/04/2009(H1N1), or wild-type A/swine/Ohio/11SW226/2011(H3N2) at a multiplicity of infection of 0.001 (for MDCK cells) or 0.1 (for A549 cells). After the cells were incubated at 37 °C for 1 h, the inocula were removed. Cells were then washed twice with PBS and incubated for 96 h at 37 °C in 5% CO₂ with 1.5 μg/mL Opti-MEM I (Life Technologies, Carlsbad, CA, USA) or Opti-MEM I containing TPCK-treated trypsin. At 12, 24, 48 and 72 h after inoculation, supernatant (200 μL) was collected from the cells and titrated, by 50% tissue culture infectious dose (TCID₅₀), in MDCK cells.
Plaque assays were performed on MDCK cells in six-well tissue culture plates. Serial dilutions were prepared from the virus stock, and 800 μL of each dilution was incubated in MDCK cells at 37 °C with 5% CO₂ for 1 h. The inocula were then aspirated, and the cells were overlaid with 2 mL of 1% agarose containing TPCK-treated trypsin (1.5 μg/mL). Cultures were incubated for 3 days at 37 °C and then fixed with methanol and stained with 1% crystal violet to reveal plaques.

U-2 OS (ATCC) and U-2 OS Flp-In T-REx Cep41-HaloTag7 (ref. ^{25 (link)}) cells were cultured in high-glucose phenol-red free DMEM (Life Technologies) medium supplemented with GlutaMAX (Life Technologies), sodium pyruvate (Life Technologies) and 10% FBS (Life Technologies) in a humidified 5% CO₂ incubator at 37 °C. Cells were split every 3–4 days or at confluency. Cell lines were tested regularly for mycoplasma contamination. Cells were seeded on eight-well glass bottom dishes (Ibidi) at three to one days before imaging. Transient transfections were performed using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s recommendations: the DNA (0.3 μg) was mixed with OptiMEM I (10 μl, Life Technologies) and Lipofectamine 2000 (0.75 μl) was mixed with OptiMEM I (10 μl). The solutions were incubated for 5 min at room temperature, then mixed and incubated for an additional 20 min at room temperature. The prepared DNA-Lipofectamine complex was added to one of the wells in an eight-well glass bottom dish with cells at 50–70% confluency. After 12 h incubation in a humidified 5% CO₂ incubator at 37 °C, the medium was changed to fresh medium. The cells were incubated under the same conditions for 24–48 h before imaging.

A reverse transfection protocol was done to deliver non-silencing negative control siRNA (scrambled siRNA) (Ambion), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or gene-specific siRNA (Ambion), into GSCs as previously described [13 (link)]. The transfection efficiency and cellular toxicity due to transfection were monitored using KDalert™ GAPDH Assay Kit (Invitrogen). Briefly, a transfection complex was prepared by diluting siRNA in 10 μl OPTI-MEMI (Invitrogen) then mixing with 10 μL OPTI-MEMI containing 0.3 uL Lipofectamine RNAi-MAX transfection reagent (Invitrogen). The siRNA transfectant was then added into each well in a 96-well plate followed by seeding 6000–9000 cells in 100 μL media to give a final siRNA concentration of 30 nM in each well. Targeted gene silencing was determined 72 h after transfection by sqRT-PCR, using a Power SYBRH Green Cells-to-CTTM Kit (Ambion).

2×10⁵ A375 or 2,5×10⁵ MeWo and 501 Mel cells/6well plate were seeded. The day after, 6ul of 20 uM siRNA stock solution (see Additional file 2: Table S5 for siRNA sequences) per well were added to 250 ul of OptiMEM I® (Invitrogen), while 10 ul of 1 mg/ml LIPOFECTAMINE 2000™ (Invitrogen) were added to additional 250 ul of OptiMEM I®. These two solutions were then combined together and the siRNA-LIPOFECTAMINE 2000™ complexes were allowed to form for 20 min at room temperature. In the meantime, the medium from each well was aspirated and replaced with 1.5 ml of fresh OptiMEM I®. The OptiMEM I®/siRNA/LIPOFECTAMINE 2000™ mixture was then added to the wells, let stand for 6 h and replaced with complete medium for 24 h (for qRT-PCR analyses) or 48 h (for western blot analyses). In alternative, at the end of the 6 h, the cells were trypsinized and used for cellular assays.