*2.9. RNA Analysis*

Total RNA was extracted from cells using the guanidinium isothiocyanate extraction method [29] after adding 20 μg of glycogen (Roche) per sample as a carrier. The content of HCV RNA, *ERLIN1* and *ERLIN2* mRNAs and *GAPDH* mRNA (for normalization) in each sample was quantitated in a two-step RT-qPCR assays using the High-Capacity cDNA Reverse Transcription Kit and the Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific). 10-fold serial dilution of plasmids containing each target sequence was used to prepare the standard curves that were used with each corresponding pair of primers: HCV (5-TCTGCGGAACCGGTGAGTA-3 and 5-TCAGGCAGTACCACAAGG-3); *ERLIN1* (5-GGGGTTGGTGGCTGTCCTGC-3 and 5-TAGCCTGGTCCACTGGGGCT-3); *ERLIN2* (5-TGTGCACACGCTTCAAGAGGTCTA-3 and 5-AATGACCAGCCCAGGGGCCA-3) and *GAPDH* (5-GAAGGTGAAGGTCGGAGTC-3 and 5-GAAGATGGTGATGGGATTTC-3). Results were normalized using *GAPDH* mRNA levels and were displayed in the figures as relative values compared to control cells.

### *2.10. Confocal Analysis*

Huh-7 cells were transfected with siRNAs and infected at high moi (moi = 3) with JFH-1 D183 virus as described above. The day before fixation cells were seeded in glass bottom 96-well plates (Thermo Scientific) for confocal analysis. At the indicated times after virus inoculation, cells were incubated in 4% paraformaldehyde for 20 min at room temperature, washed 3 times with PBS and processed for immunofluorescence analysis and LD staining as previously described [38]. In brief, fixed cells were first incubated for 1 h at room temperature in blocking bu ffer (1xPBS, 10% FBS, 3% BSA, 0.3% Triton X-100), washed 3 times with PBS and then incubated for 1 h with a mixture of primary antibodies at the appropriate concentrations that were prepared in binding buffer (1xPBS, 3% BSA, 0.3% Triton X-100). The primary antibodies used for immunofluorescence were: a purified mouse monoclonal anti-core (clone C7-50) at 1:200 dilution and a rabbit polyclonal anti-NS5A (MS5) at 1:1000 dilution. After 3 washes with PBS, cells were incubated for one more hour in binding buffer containing a mixture of fluorophore-conjugated secondary antibodies at 1:1000 dilution (Invitrogen) and Hoechst dye (Invitrogen) for nuclei staining. LD staining was performed by incubating the cells for 30 min at room temperature with the LipidTox reagen<sup>t</sup> (Invitrogen) at 1:500 dilution in PBS. Images of 1024 × 1024 pixels at 8-bit gray scale were acquired with a 40× objective (pixel size 0.345 μm) on a LSM 710 confocal laser scanning microscope (Zeiss, Dublin, CA, USA). All images were taken with exactly the same settings. Fiji/ImageJ software analysis package [39] was used to quantitate the fluorescence intensity signals in each image.
