Turbofecttm

Lyophilized FITC-labelled RNA duplexes (Dharmacon Thermo Scientific) were obtained in 2’ deprotected, annealed and desalted form, dissolved in PCR grade water (Roche) at 3 µg/µl and stored in aliquots at −80°C. The transfection solution was 1 µg/µl siRNA, 10% polyethylene glycol (PEG) (Carbowax 6000, Union Carbide) and 20% TurbofectTM (Thermo Fisher Scientific, Catalog # R0541). For 2.8 µl of siRNA preparation, 1 µl of siRNA, 1.2 µl of 20% PEG stock and 0.6 µl of TurbofectTM were incubated at 21°C for 30 min before application. This technique was also tested using pCAβ-EGFPm5-mU6 (Bron et al., 2007 (link)), a kind gift of Dr. Matthieu Vermeren (Department of Physiology, Development and Neuroscience, University of Cambridge, UK). The final transfection solution contained 2 µg/µl plasmid, 10% PEG and 40% TurbofectTM. For a final 5 µl of solution, 1 µl of plasmid, 2 µl of 20% PEG stock and 2 µl of TurbofectTM were used; this solution was only used once. For siRNA delivery in ovo, borosilicate glass capillaries (WPI, outside diameter 1.5 mm, inside diameter 1.12 mm) were pulled on a Narishige Puller PC-10 at 62°C. Tips were broken to obtain a suitably narrow internal diameter and capillaries attached to a rubber tube/mouth-pipette.

Ba/F3-gp130 cells were retrovirally transduced using pMOWS plasmids coding for different SyCyRs as described previously [2 (link)]. The packaging cell line was Phoenix-Eco. After transduction cells were grown as described above and supplemented with puromycin (1.5 μg/ml) and/or hygromycin B (1 mg/ml) (Carl Roth, Karlsruhe, Germany). CHO-K1 cells were stably transfected with TurboFect^TM (Thermo Fisher Scientific, Waltham, United States) and then selected using 1.125 mg/ml G-418 sulfate (Genaxxon, Ulm, Germany).

HeLa Kyoto or HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum (FBS) (Sigma), 2 mM GlutaMax-I (GIBCO), 50 U/ml penicillin, and 50 μg/ml streptomycin (GIBCO). Plasmids for transfection were prepared using a Plasmid Miniprep purification kit (Evrogen, Russia). Transfection was performed using TurboFectTM (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

All chemicals used were of analytical grade. Restriction endonucleases, T4 DNA polymerase, Taq polymerase and T4 DNA ligase were obtained from MBI Fermentas. Chemicals 12-O-tetradecanoylphorbol 13-acetate (TPA) and MG132 were from Sigma-Aldrich (Stockholm, Sweden). TurboFectTM and Lipofectamine™ transfection reagents were purchased from Thermo Fisher Scientific (Uppsala, Sweden). Horseradish peroxidase-linked anti-immunoglobulins were from Dako A/S (Sundbyberg, Sweden). The monoclonal antibody C7-50 [34 (link)] recognizing HCV C was from Santa Cruz Biotechnology (Heidelberg, Germany).

To monitor promoter activity, cells were transiently transfected with indicated pcDNA3.1-E2F1 plus 1 µg pGL3-SLC16A1-AS1-Prom using Turbofect^TM (Thermo Scientific) according to the instructions. Luciferase activity was measured 36 hours after transfection using the Luciferase Reporter Assay System (Promega). Samples were normalized to total protein concentration in cell extracts. For mutation studies, 1×10⁵ UMUC-3 or RT-4 cells were transiently transfected with 0.4 µg or 0.8 µg pGL4-SLC16A1-AS1-Prom (BS full or BS deletion), 0.2 µg or 0.4 µg pGL4[hRluc75], 0.2 µg or 0.4 µg pcDNA3.1-E2F1 and/or pGE-SLC16A1-AS1 using Neon Transfection System (Invitrogen) according to the instructions. Luciferase activity was measured 72h post-transfection using Dual-Luciferase Reporter Assay System (Promega). Firefly-Luciferase activity (pGL4-SLC16A1-AS1-Prom) was normalized to Renilla Luciferase activity (hRLuc).

HeLa Kyoto or HEK293T cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum (FBS) (Sigma), 50 U/ml penicillin, and 50 μg/ml streptomycin (GIBCO). Plasmids for transfection were prepared using a Plasmid Miniprep purification kit (Evrogen, Russia). Transfection was performed using TurboFectTM (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

Dissected somite strips were collected in a 2 ml LoBind tube (Eppendorf) containing L15 medium, and sclerotome cells were dissociated with a 25G needle, after which 20 μl of cells were transferred into each chamber of a 4-chambered cell culture slide (BD Falcon) containing 490 μl medium per chamber pre-warmed at 37°C. To maintain sclerotome differentiation a notochord fragment was added to each well. Slides were cultured in a humidified box at 37°C for 16 hr, after which csPDI was assessed by anti-PDI- and PNA-staining as described above for somite strips. For retinal cells, eyes were removed from stage 22–24 embryos using a microscalpel, and retinal cells dissociated and stained as for sclerotome cells. For siRNA transfection, cells were incubated at 38°C for 16 hr. 10 μl of transfection mix [12.5 μg siRNA in 100 μl 5% glucose and 1.5 μl TurbofectTM (Thermo Fisher Scientific)] in 490 μl of DMEM (Sigma-Aldrich) supplemented with B-27 (Life Technologies) and NGF (Sigma-Aldrich) was then added to cultures. After overnight incubation at 38 °C cells were washed x3 in DMEM and incubated for 3 hr with B-27/NGF-supplemented DMEM.