p21 siRNA (h)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-80 pmol. Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 1–3 days at 37°C.

Protocol tips
siRNA concentration-80 pmol. Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 1–3 days at 37°C.

Publication protocol

The COV434 cell line was a kind gift from Sandrine Caburet. COV434 were cultured in DMEM/F12 medium (ThermoFisher, Waltham, MA, USA) supplemented with 10% FBS (ThermoFisher), 1% GlutaMAX (ThermoFisher), 100 μg/ml penicillin and 100 U/ml streptomycin (ThermoFisher) at 37 °C, 5% CO2. COV434-Mock and COV434-FGF1 were obtained following stable transfection with 2.5 µg of pcDNA3.1D V5-His plasmid containing or not the FGF1 coding sequence, using 8 µl of Lipofectamine LTX and 2.5 µL Reagent plus (Life Technologies, Carlsbad, CA, USA). Transfected cells were selected in 500 µg/mL of Geneticin G418 (Life Technologies) for two weeks. The isolated clones were amplified and used for experiments. For p21 and p53 knockdown experiments, 3 × 105 cells were plated on 6-well plates. At 30% confluence, siRNA transfection was done using 80 pmol of human p21 siRNA, human p53 siRNA or siRNA-A control (Santa Cruz, Dallas, TX, USA) and 4 µl lipofectamine RNAimax (Invitrogen). Etoposide (Sigma-Aldrich, St Louis, MO, USA) was used at 25 µg/mL. Pifithrin-α and pifithrin-µ (2-phenylethynesulfonamide, PFTμ) are from Enzo Lifesciences Farmingdale, NY, USA, and FGFR1/3 inhibitor from Tocris Bioscience, Bristol, UK. Recombinant FGF1 (R&D Systems, Minneapolis, MN, USA) was used in combination with 10 µg/mL heparin (Sigma-Aldrich).

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Manufacturer protocol

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