24 well cell culture plates

Antennae of both 14 dpi and similarly aged non-infected adult females were collected during three hours within the activity peak. Individuals were cold-anaesthetized, the antennae removed using forceps, and then immediately transferred into 24-well cell culture plates (Sarstedt, Germany) containing 500 μl RNAlater (Thermo Fisher Scientific, Sweden). Both antennae of the same individual were placed in the same well. All samples were stored at room temperature for 24 h, and then transferred to -80°C until RNA extraction. Simultaneously, whilst dissecting the antennae, the bodies of infected females were placed in separate wells in 24-well plates and subsequently subjected to qPCR analysis to quantify viral load, as described above. RNA extraction and DNase I digestion were performed using the RNeasy Mini Kit (Qiagen, Sweden) following the manufacturer’s protocol, on the antennae of successfully infected females. Aliquots of RNA were tested for both quantity and quality on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Following the quantification of viral load (above), antennal RNA samples of successfully infected individuals were pooled in tubes containing a total of 50 antennae, according to the level of infection, and stored at -80°C.

Sulfuric acid (98% for analysis), aqueous hydrogen peroxide (30% for synthesis), benzene (ACS, ISO, Reag. Ph. Eur. for analysis), dichloromethane (DCM; ACS, ISO, Reag. Ph. Eur. for analysis), ethanol (>96% not denaturized), and chloroform (ACS, ISO, Reag. Ph. Eur. for analysis) were purchased from VWR international^® (Darmstadt, Germany) and 3-iodopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane (97%) and (N-methylaminopropyl)trimethoxysilane from abcr^® (Karlsruhe, Germany). Poly(ethylene oxide) (PEO4600, nominal M_W = 4600 g/mol), N,N,N′,N′-tetramethylethylenediamine (TMEDA) (ReagentPlus^®, 99%, freshly distilled), 3-sulfopropylmethacrylate potassium salt (SPM) (98%), triethylamine (≥99%) and 2M lithium diisopropylamine (LDA) in tetrahydrofurane (THF), N-propyl gallate and α-bromoisobutyryl bromide (98%) were purchased from Sigma Aldrich^® (Taufkirchen, Germany). HL5c medium was obtained from Formedium (Hunsanton, UK), 24 well cell culture plates from Sarstedt (Nümbrecht, Germany), glutaraldehyde (0.5%) from Plano (Wetzlar, Germany), and the silicon wafer with diameter of 150 mm and <100> orientation from SI-MAT (Kaufering, Germany). All chemicals were used as received.
Ultrapure water with a resistivity of 18.2 MΩ∙cm was obtained fresh from an ELGA Pure Lab ultra machine (Celle, Germany). Dry solvents were prepared according to previously published procedures [17 ].

For the overexpression experiments, pCMV6 entry vectors containing the open reading frames (ORF) of the five candidate mouse proteins mOCIAD1, mPAFAH2, mHTATIP2, mPDCD6 and mSAR1B were purchased from Origene Technologies GmbH (Herford, Germany). To equip the proteins with C-terminal myc-tags, the ORFs were amplified by RT-PCR, using gene-specific primers equipped with appropriate restriction cites (see Table S3), and recloned into pCMV3A mammalian expression vectors (Agilent Technologies, Santa Clara, CA, USA). The correct insertion and preservation of the ORF was subsequently checked by sequencing (Eurofins Genomics, Ebersberg, Germany).
For the colocalization experiments, HepG2 cells and mouse embryonic fibroblast (for preparation see [18 (link)]) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% penicillin/streptomycin. For immunofluorescence microscopy, the cells were seeded into 24-well cell culture plates (Sarstedt, Nümbrecht, Germany) equipped with Poly-D-lysine-coated (Thermo Fisher Scientific, Waltham, MA, USA) glass coverslips. For overexpression experiments, the cells were transfected on the next day with LipofectamineTM3000 reagent (Thermo Fisher) containing 0.5 μg plasmid DNA per well. After 24 h of incubation, the cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 min, at RT.