Siport

siRNAs against BCA3 (C11orf17) were purchased from Ambion (Thermo Fisher Scientific, s.r.o., Prague, Czech Republic). One day before transfection, HEK 293 cells were seeded in 12-well plate at the density 3 × 10⁵ cells/mL. Next day the cells were transfected with siRNA at various concentrations using siPORT (Ambion), according to the manufacturer’s instructions. At 24 h post the first transfection, the cells were transfected with HA-BCA3 expression vector (0.4 µg per well) using Fugene. At 24 h post-second-transfection, the cells were harvested and analyzed for presence of HA-BCA3 by Western blot analysis. In an experiment when the effect of BCA3 knockdown on HIV-1 release was analyzed, HEK 293 cells were grown in 60 mm plates and the transfection procedure was carried out as described above, with the exception that the psPAX2 vector was cotransfected with that encoding HA-BCA3. At 24 h post-second-transfection, the culture media were filtered through 0.45 µm filter and ultracentrifuged through a 20% sucrose cushion at 40,000 rpm for 1 h in a SW41 rotor. Both the viral pellet and producing cells were analyzed by Western blot and quantification of virus release was determined as a ratio of protein bands’ intensity of virion-associated CA to cell-associated CA by using Fusion CAPT Advance software (Vilber Lourmat, Marne-la-Vallée, France).

GBM 28, SJ-GBM2 and U87MG cells were transfected with Akt siRNA (Cell Signaling Technologies, Danvers, MA). Transfection was performed using siPORT (Ambion Inc, Austin, TX) transfection reagent and as per manufacturers protocol. Cells were transfected with 100 nM Akt siRNA and after 12 h post transfection, cells were treated for additional 48 h with 5 μM penfluridol. The cells were collected after treatment and processed for western blot analysis as described by us before [33 (link)].

GBM28, SJ-GBM2 and U87MG cells were transfected with GLI1 siRNA (Santa Cruz Biotechnology, Inc, Dallas, TX) using siPORT (Ambion Inc, Austin, TX) transfection reagent as per manufacturer's protocol. Cells were transfected with 100 nM GLI1 siRNA and after 12 h post transfection, cells were treated for additional 48 h with 5 μM penfluridol. The cells were collected after treatment and processed for western blot analysis as described by us before [20 (link)].

For luciferase assays, the regions of 3′UTR of FoxO1 and CDKN1B containing the miRNA binding sites were cloned in pGL3 control vector (Promega, Milan, Italy) at XbaI restriction site, downstream luc gene. The 3′UTR regions of target genes were amplified from human blood cells genome by PCR reaction using the following primers:
primer forward FoxO1: CCCAATGTGTGCAGGT TATG; primer reverse FoxO1: AGGTCCAAGGCTGT TCAATG; primer forward CDKN1B: TTCATGGAATGG ACATCCTGT; primer reverse CDKN1B: CCTTCCCCAA AATTGCTTCT.
After cloning in DH5α bacteria strain, positive clones were identified by PCR on colonies using the same forward oligonucleotides described before and a reverse oligonucleotide that aligns on pGL3 control vector. Clones that were positive for PCR were screened also for digestion with XbaI. Only clones that were positive for both screenings were sequenced. Deletion of the seed sequence was performed by PCR.
These plasmids were used to transfect HEK293 cells with siPORT (Ambion Life Technologies, Paisley, UK) in the presence of mature microRNA-196a to evaluate the true bind of this miRNA to its own site on 3′UTR region of predicted target genes. Luciferase activity was assayed with a dual luciferase assay system (Promega, Milan, Italy) as described in the manufacturer instructions.

Cultured chicken PGCs were transfected with inhibitors against the gga-miR-302b-5P and gga-miR-302b-3P at 100 nM final concentration using the transfection agent siPORT (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). We used the following inhibitors given in Table 3 below.

Two weeks before the transfection, chicken FS101 and FS111 PS PGC lines were thawed. After two weeks in the culture, PGCs were collected by centrifugation and plated to 96-well plates (1000 cells/well). Next day, the cells were transfected with anti-miR-302b-5P and anti-miR-302b-3P vectors (at 100 nM final concentration) (Supplementary Table 1) (Applied Biosystems, Life Technologies, Carlsbad, US) using a siPORT™ (Applied Biosystems, Life Technologies, Carlsbad, US) transfection agent according to the manufacturer's instructions. Three 96-well plates (as biological replicates), with 6-6 parallel wells, were prepared for each condition. The proliferation rate of treated and control cells was measured using CCK-8 reagent (1 : 10, Dojindo Laboratories, Japan) every day, for 3 days. For detailed RNA expression analysis, cells were harvested in lysis buffer of RNAqueous®-Micro Kit (Applied Biosystems, Life Technologies, Carlsbad, US) 48 hours following the transfection.