Mission esirna

HLFs were seeded at 10 × 10⁴ cells/cm² and transfected with 60 pmol of endoribonuclease prepared siRNA (esiRNA) against ELAVL1 (MISSION^® esiRNA, Sigma, St. Louis, MO, USA) or non-targeting control esiRNA (MISSION^® esiRNA, Sigma) with Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. One hour after the transfection, 10% MEM medium was added on the cells. After 24 h, the cells were switched to serum-free MEM for 44 h, after which cellular proteins were collected. Confirmation of HuR knockdown was done by Western blot within 68 h after transfection.

FTC-133 cells were used to study the effect of PROX1 knock-down on hallmark characteristics of malignant cells: migration, invasion, motility, proliferation, apoptosis and anchorage–independent growth. Cells were grown to 60-70% confluence in 24-well or 6-well plates and transiently transfected with either non-targeted pool of small interfering RNAs (MISSION^® siRNA Universal Negative Control #1, Sigma-Aldrich, St. Louis, MO, USA), a pool of specific siRNAs against human PROX1 (MISSION^® esiRNA, Sigma-Aldrich, EHU053851) or with PROX1 siRNA (h) (sc-106451, Santa Cruz Biotechnology, USA), using Lipofectamine^® 2000 Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ protocol. The optimal transfection efficiency was obtained using a cell density of 4.5x10³ per well, 30 nM siRNAs in OptiMem medium (Roche, Basel, Switzerland), and 1.5 μl Lipofectamine/OptiMem. Cells were incubated with the transfection mix for 48 hours and then harvested for RNA purification or used for other assays. The experiments were conducted on at least three different cell passages and run in triplicate (three samples of the same cell passage).

Lentiviral shRNAs targeting AHR and non-targeting control were obtained from Sigma-Aldrich (St. Louis, MO, USA). Following transduction, H1975 cell lines were selected with 0.5 µg/ml puromycin for 7 days. Clones were established from the transduced populations using limiting dilution. After lentiviral transduction of cells with EGFP-ffluc-epHIV7 vector, GFP-positive cells were isolated using fluorescence-activated cell sorting.
For transient knockdown of ATF4, specific MISSION® esiRNA (Sigma-Aldrich, Munich, Germany) and MISSION® siRNA Universal Negative Control were introduced by RNAiMAX (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
For sequences of shRNAs and siRNAs see Supplementary Table 2.

MSCs were seeded at 60,000 cells cm⁻² in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose content, 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂. After 24 h, cells were washed with Opti-MEM reduced serum medium (ThermoFisher) and transfected using pre-designed MISSION esiRNA (Sigma-Aldrich) against mouse NaBC1 transporter in X-tremeGENE siRNA Transfection Reagent (Roche), following manufacturer’s instructions. MISSION siRNA Fluorescent Universal Negative Control 1, Cyanine 3 (NC, Sigma-Aldrich) was used as a control of transfection efficiency.

The siRNA targeting TRPV1 was purchased from Sigma-Aldrich Inc. MISSION esiRNA (human TRPV1) (EHU073721). Scrambled siRNA was used as negative control. In brief, once the seeded mCRC cells had reached 50% confluency, the medium was replaced with Opti-MEM, the serum in the medium reduced without antibiotics (Life Technologies, Milan, Italy). siRNAs (100 nM final concentration) were diluted with Opti-MEM I reduced serum medium and mixed with Lipofectamine™ RNAiMAX transfection reagent (Life Technologies, Milan, Italy) pre-diluted in Opti-MEM), according to the manufacturer’s instructions. After 20 min incubation at room temperature, the mixtures were added to the cells and incubated at 37 °C for 5 h. Transfection mixes were then completely removed and fresh culture media was added again and silenced cells were used 48 h after transfection. The effectiveness of silencing was determined by immunoblotting (see Figure S2).

For ODZ1 knockdown experiments, we used two different shRNAs with sequences 5′-AATGGAGAATACGAGAAAGGACA-3′ and 5′-AAGACCGACATCTATGGACAGAA-3′ and a scrambled shRNA (Cat. No. P100042) as a negative control (all from Vigene Biosciences, Rockville, MD). Cells were transfected with shRNAs by nucleofection. Stat3 was silenced by transfecting GBM cells with a mixture of Stat3-specific (Cat. No. EHU122051) or negative control (Cat. No. EHUEGFP) siRNAs (MISSION esiRNA, Sigma-Aldrich) using Lipofectamine RNAiMAX (Invitrogen).

C2C12 were seeded at confluence density (20,000 cells/cm²) in growth medium. After 24 h cells were transfected with MISSION esiRNA (Sigma-Aldrich) in X-tremeGENE siRNA Transfection Reagent (Roche), following manufacturer’s instructions. Cell transfection was carried out in Opti-MEM Reduced Serum medium (Thermofisher). MISSION siRNA Fluorescent Universal Negative Control 1, Cyanine 3 (NC. Sigma-Aldrich) was used as transfection control. Transfected myoblasts were cultured for 3 days with differentiation medium (DMEM/2% FBS/1% P/S). Then, myogenic differentiation was assessed by immunofluorescence of MHC.