Metafectene reagent

The experimental setup to perform replication-independent, transient polyprotein expression has been described [43 (link)]. Viral polyproteins were expressed from pcite NS3-NS5B encoding HCV nonstructural proteins NS3-NS5B of genotype 1b (Con1) or genotype 2a (JFH1), respectively. Briefly, for SDS-PAGE and Western blot analysis of viral proteins 2 x 10⁶ Huh-7/T7 cells were infected with MVA-T7^pol vaccinia virus [110 (link)] and subsequently transfected with 8 μg of the respective plasmid DNA by using a PEI transfection protocol. Additional infection of Huh7-T7 cells with MVA-T7 was used to boost the expression of T7 RNA polymerase to allow efficient replication-independent expression of the HCV polyprotein. In the case of IF analysis for protein localization Huh7-Lunet/T7 cells were transfected with 8 μg of the indicated plasmid DNA by Metafectene reagent (Biontex Laboratories GmbH, Munich, Germany) without prior MVA-T7pol vaccinia virus infection.

NG108-15 neuroblastoma cells were purchased from Sigma Aldrich and grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 100 μg/ml streptomycin and 100 units/ml in penicillin in a 5% CO₂atmosphere at 37°C. For live cell imaging studies, cells were seeded on glass coverslips, coated for 3 h with laminin (50 μg/ml, L2020 Sigma) in 12-multiwell plates. Cells were transfected 24 h later with intermolecular RhoA/Cdc42 FRET sensors (Murakoshi et al., 2011 (link)) using Metafectene reagent (Biontex Laboratories) following the manufacturer's protocol and imaged 1 day after transfection. mEGFP-RhoA-C1 (Addgene plasmid #29674), mEGFP-Cdc42-C1 (Addgene plasmid #29673), mCherry-Rhotekin(8–89)-mCherry-C1 (Addgene plasmid #29675) and mCherry-Pak3(60–113)/S74A/F84A-mCherry-C1 (Addgene plasmid #29676) were a gift from Ryohei Yasuda (Murakoshi et al., 2011 (link)).

The applied procedures have been described [35 (link)]. HCV nonstructural proteins were expressed from pcite_ plasmids. Briefly, 2 x 10⁶ Huh7/T7 cells were infected with MVA/T7^pol vaccinia virus [36 (link)] for 1 h at 37°C and subsequently transfected with 4 or 8 μg of plasmid DNA by using Metafectene reagent (Biontex Laboratories GmbH, Munich, Germany). Transfected cells were incubated for either 24 h or 48 h at 37°C for protein expression. Cells were harvested at indicated time points and subsequently lysed for SDS-PAGE and western blot analysis.

Freshly isolated FAPs were cultured at 37°C in alpha-MEM (Hyclone) supplemented with penicillin (100 U/mL), streptomycin (1 mg/mL), and 20% FBS (Hyclone). HEK293T cell line (ATCC) was grown at 37°C in DMEM (Hyclone) supplemented with antibiotics and 10% FBS. The cell line was tested for mycoplasma contamination. All transfections were performed using polyethyleneimine (PEI; Polysciences) and Metafectene reagent (Biontex) following the manufacturer’s instructions. Full-length and point-mutated constructs of SMN were subcloned into the pcDNA4-HisMax vector. N-terminal flag-tagged full-length, deleted, and point-mutated construct of mouse Bap1 and human BAP1 were subcloned into the pcDNA4 or pcDNA3.1 vector (Addgene). HA-tagged Ub was subcloned into pcDNA3 vector.

TMSCs were transfected with siRNA for PCOLCE2 (50 nM, Invitrogen, Carlsbad, CA, USA) using Metafectene reagents (Biontex Laboratories GmbH, Munich, Germany). After 72 h, cells were utilised for further study. The sequences of predesigned Stealth Select siRNA (Thermo Fisher Scientific, Waltham, MA, USA) against PCOLCE2 are as follows: sense, 5′-UGGUGAUAGUCCACCUGCGCCAAUU-3′; antisense, 5′-AAUUGGCGCAGGUGGACUAUCACCA-3′.