Knockdown of neogenin blocks HJV release but does not affect HJV cell surface expression. We used siRNA specific to human neogenin and Lipofectamine RNAiMax to knockdown the endogenous neogenin in control and HJV-HepG2 cells as described under “Experimental Procedures.” Scrambled siRNA was used as a parallel negative control. A, cell-associated protein. PI-PLC digestion was used to examine the effect of neogenin knockdown on the dynamic HJV expression on cell surface. The cells from above were incubated in the presence or absence of PI-PLC at the concentration of 1 unit/ml for 2 h in 37 °C CO₂ incubator. HJV, neogenin (neo), and actin in cell lysates (L) and the HJV in supernatant (HJV (sup)) were detected by Western blot analysis. B, HJV release. At 24 h after the second transfection of cells with siRNA, the cells were pooled and subcultured into 12-well plates with complete medium containing 10% fetal calf serum. Approximately 48 h later, 100 μl of the CM was subjected to SDS-PAGE and Western blot analysis for HJV (HJV (CM)). C, flow cytometry analysis of the cell surface HJV. Flow cytometry analysis of cell surface HJV was analyzed as described under “Experimental Procedures” and expressed as arbitrary units (a.u.). The averages of four individual analyses and the standard deviations were presented. All of the other experiments were repeated at least three times with consistent results.

HJV release is correlated with neogenin degradation. A, expression of HJV, but not G320V mutant, decreases the level of neogenin protein in HepG2 cells. Cell lysate from 2 × 10⁵ cells and CM from 0.8 × 10⁵ cells of overnight culture were subjected to SDS-PAGE, followed by immunodetection of the HJV in both cell lysates (HJV (L)) and CM (HJV (CM)), and neogenin (neo (L)) and actin in the lysate. B, PI-PLC digestions. HJV, G320V, and control-HepG2 cells in 12-well plates at approximate 70% confluence were incubated in 300 μl of MEM with or without the addition of PI-PLC (1 unit/ml) for 3 h at 37 °C CO₂ incubator. Approximately half of the cell lysate or supernatant were subjected to SDS-PAGE, followed by immunodetection of HJV in both cell lysate (L) and supernatant (sup), and neogenin (neo) and actin in the lysate. C, bafilomycin A increases cellular neogenin. Control (C) and HJV-HepG2 cells in 12-well plate were incubated for 4 h in complete medium with or without the addition of 100 nm bafilomycin A (Baf). Neogenin, HJV, and actin in cell lysate (L) and HJV in approximately one-third of CM were detected by Western blot using the specific antibodies. All of the experiments were repeated at least three times with consistent results.

HJV undergoes endocytosis. A, internalization of biotinylated cell surface HJV. Cell surface proteins in control (C) and HJV-HepG2 cells were biotinylated at 4 °C, followed by incubation at 37 °C for 0 and 10 min (0′ and 10′). After stripping the biotin remaining on cell surface, the internalized HJV (intrnlzd) was isolated using streptavidin-agarose beads and subjected to Western blot. Approximately 10% of lysate from the cells without biotinylation (lysate) and the total biotinylated cell surface HJV without incubation and stripping (cs) were included for the analysis. B, filipin inhibits HJV internalization. The experiments were conducted essentially the same as described in A, except that the biotinylated cells were first preincubated in the absence or presence of 10 μg/ml filipin or 160 μm dynasore (dynas) at 4 °C for 30 min before the incubation at 37 °C.

The ectodomain of neogenin blocks HJV release. HFE2 stably transfected (HJV) and empty vector transfected control (C)-HepG2 cells in 12-well plate were incubated in the absence or presence of the soluble ectodomain of neogenin at 0-1000 nm for 24 h. Approximate half of the cell lysate (L) and 20% of the CM was subjected to Western blot analysis for HJV and neogenin (neo). Actin was also probed in the lysate as a protein loading control. The experiments were repeated three times with consistent results.

Depletion of cholesterol inhibits HJV release. A, dynasore does not inhibit HJV release. HJV-HepG2 cells in 12-well plates were incubated in the presence of 0, 80, and 160 μm of dynasore for 2 h at 37 °C. HJV in both cell lysate (L) and 50% of CM was detected by Western blot. B, ¹²⁵I-Tf-uptake. HepG2 cells in 6-well plates with approximate 80% confluence were first preincubated in absence or presence of dynasore (80 and 160 μm; D80 and D160, respectively) and filipin (10 μg/ml) (F) for 30 min at 37 °C. ¹²⁵I-Tf uptake was initiated by incubating the cells in the presence of 50 nm ¹²⁵I-Tf and the same concentrations of inhibitors. After 8 min of incubation at 37 °C, membrane-bound ¹²⁵I-Tf was removed by acid wash. The ¹²⁵I radioactivity was counted. The inclusion of 1 mg/ml unlabeled cold Tf was used as the nonspecific uptake control. The rates of specific uptake in the presence of inhibitors were expressed as the percentage of the corresponding controls. The results are from four individual experiments. ^*, p = 0.0281; ^**, p < 0.0001. C, wild type and K44A mutant dynamin. HJV-HepG2 cells in 12-well plates were infected with the mixture of tTA activator virus and different amounts of adenovirus containing either wild type or K44A mutant dynamin (0, 0.048, 0.096, 0.19, and 0.38 μl of stock virus/well). tTA activator virus alone was used as a negative control. The cells were first incubated for ∼18 h to allow the expression of introduced dynamin. Afterward, ∼20% of the CM collected from the incubation between 18 and 42 h post-infection was detected for HJV by Western blot. In addition, dynamin (dyn), HJV, and actin in the cell lysate (L) at 42 h post-infection were also analyzed. D, filipin inhibits HJV release. HJV-HepG2 cells in 12-well plates were incubated in the presence of 0, 1, 5, and 10 μg/ml of filipin for 2 h at 37 °C. HJV in both cell lysate (L) and 50% of CM was detected by Western blot. E, filipin inhibits the biotinylated cell surface HJV release. Cell surface proteins in HJV-HepG2 cells were biotinylated at 4 °C, followed by incubation in the presence of 0, 10, and 20 μg/ml of filipin at 37 °C for 2 h. The biotinylated HJV that was released into the medium (HJV (CM)), as well as the total biotinylated cell surface HJV (T) (HJV (S)), was isolated using streptavidin-agarose beads and subjected to Western blot (lower panel). The total HJV in cell lysate (L) was also detected (upper panel). All of the experiments were repeated at least three times with consistent results. In each experiment, the lysate and CM from control-HepG2 cells (C) were included as a negative control for HJV.