Characterization of matriptase-2–mediated HJV cleavage. (A) Half-life of matriptase-2 protein. HepG2 cells stably expressing matriptase-2 (HepG2-M2) were incubated with 100 μg/mL cycloheximide (CHX) for the indicated time intervals. Cell lysates (L) were then prepared and analyzed by immunoblotting by the use of anti–matriptase-2 (MT2) or anti–β-actin (actin) antibodies. HepG2 cells stably transfected with a pcDNA3.1 empty vector (C) were included as a negative control for matriptase-2 (top). The intensities of the bands were quantified with fluorescent secondary antibodies and the Odyssey Infrared Imaging system. The relative fractions of matriptase-2 remaining in cell extracts at each time point were calculated. The graph was generated from 4 separate experiments with duplicates for each time point (bottom). Error bars represent the SD. The estimated half-life of matriptase-2 was approximately 2.5 hours. (B) Matriptase-2 cleaves cellular HJV in HEK293 cells. HEK293 cells stably expressing HJV (HEK293-HJV) in 12-well plates were transiently transfected with an increasing amount of either pcDNA3.1-TMPRSS6 (M2) or a pcDNA3.1-TMPRSS6 construct with a C-terminal myc tag (M2 myc) at 0, 0.13, 0.33, 0.66, 1.33, or 2.0 μg DNA with Lipofectamine 2000. In the mean time, a decreasing amount of pcDNA3.1 empty vector (2.0, 1.87, 1.67, 1.34, 0.67, or 0 μg DNA) was also simultaneously introduced to allow an equal transfection. Approximately 24 hours after transfection, culture medium was replaced with fresh complete medium (Dulbecco modified Eagle medium [DMEM]/10% fetal calf serum [FCS]). At approximately 56 hours after transfection, CM was collected, and cell lysate was prepared. The total lysate and 10% of CM were subjected to SDS-PAGE, followed by immunodetection of myc, matriptase-2 (M2), HJV, and β-actin (actin) in the lysate (L) and HJV in CM. Chemiluminescence was used to visualize the bands. HEK293 cells stably transfected with pcDNA3.1 (C) were included as a negative control for HJV. Experiments were repeated 4 times with consistent results. (C) Coculture of HepG2-M2 cells with HEK293-HJV (HEK-HJV) cells. HepG2 cells (HepG2-C) alone, HepG2-M2 cells alone, HEK293-HJV cells alone, HepG2-C + HEK293-HJV cells, and HepG2-M2 + HEK293-HJV cells were seeded in 12-wells plates at 2.0 × 10⁵ cells per cell type in 1.5 mL of DMEM/10% FCS. Approximately 24 hours after plating, culture medium was replaced with 1.5 mL of fresh DMEM/10% FCS. After another 24 hours of incubation, CM was collected and cell lysate was prepared. Total lysate and 10% of CM were subjected to SDS-PAGE. Matriptase-2 (M2), HJV, and β-actin (actin) in the whole lysate (L) and HJV in 10% of CM were immunodetected as described previously. HEK293-HJV cells transiently transfected with pcDNA3.1-TMPRSS6 (pcDNA3-M2) were included as a control (lanes 11 and 12). Experiments were repeated 6 times with consistent results. (D) Matriptase-2 knockdown. SMARTpool siRNA specific for human TMPRSS6 (Dharmacon) was used to knock down the matriptase-2 in HepG2-M2 cells (lanes 9 and 10). Scrambled siRNA served as a negative control (lanes 7 and 8). RNAiMAX reagent (Invitrogen) was used for the transfection. siRNA transfection was conducted in 12-well plates in complete medium. Approximately 24 hours after siRNA transfection, HJV was introduced by the use of FuGene HD transfection reagent (Roche). Approximately 72 hours after siRNA transfection, CM was collected and cell lysate was prepared. The total lysate and 10% of CM were subjected to SDS-PAGE. Matriptase-2 (M2), HJV, and β-actin (actin) in the lysate (L) and HJV in CM were immunodetected as described previously. HepG2 cells (HepG2-Ctrl) transiently transfected with pcDNA3-HJV were included as a control (lanes 3 and 4). Experiments were repeated 4 times with consistent results.