Netrin-1 impedes EGFR recycling.

Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A. Li diagrams and Li coefficient calculations (B) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y-axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software (http://rsb.info.nih.gov/ij/plugins/track/jacop.html). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment (n = 2). C. Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D. Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in (C) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data.