Figure 1.
Presence of N-terminal gelsolin fragments in hepatocyte conditioned medium.
Panel A: Western blot of CM with chicken antibodies raised against peptides (G1 and G2) selected from the N-terminal region of gelsolin. Purified gelsolin from human plasma was included as a positive control in the experiment. SAGM medium, used for culture of stellate cells, was used as a negative control. (B) Immunoprecipitation of CM with healthy control and chronically HCV infected patient sera, followed by Western blotting with chicken anti-gelsolin antibody G1. The positions of protein molecular weight markers (kD) are shown on the left.
Figure 2.
Effect of CM from Huh 7 cells transfected with gelsolin fragments on LX2 growth.
Panel A: CM from Huh7 cells transfected with plasmid constructs- D2 encoding N-terminal half (amino acids 1–450) and D3 encoding C-terminal half (amino acids 450–782) displayed distinct LX2 cell viability. LX2 cells treated with IHH CM as a positive control and untreated LX2 as a negative control were included in the experiment. Cell viability was determined from triplicate culture wells by a CellTiter 96 Aqueous non-radioactive cell proliferation assay (Promega) at different time points and presented as mean and standard deviations. Panel B: LX2 cell viability following incubation with CM from Huh7 cells transfected with deletion mutants encoding gelsolin fragments (amino acids 1–450, 1–70, 1–130 and 70–450) were similarly determined. Effect of medium (untreated) and IHH CM treated positive controls were included for comparison. Cell viability was determined from triplicate culture wells by a CellTiter 96 Aqueous non-radioactive cell proliferation assay (Promega) at different time points and presented as mean and standard deviations. Panel C: Expression of gelsolin N-terminal 1–70 fragment in CM from 293T cells transfected with FLAG-tagged D2–F1 construct. CM from transfected cells was immunoprecipitated with anti-FLAG antibody and analyzed by Western blot using anti-gelsolin G1 antibody. CM from mock-transfected 293T cells served as negative control.
Figure 3.
Antibody to N-terminal fragment of gelsolin antagonizes LX2 cell death.
CM from IHH (panel A) or from Huh 7 cells transfected with N-terminal gelsolin fragments consisting of amino acids 1–70, 1–130 and 70–450 (panel B) was preincubated with immobilized gelsolin G1 or TRAIL antibody, and subsequently tested for LX2 cell growth. Effect of normal medium (untreated) and CM without preincubation were included for comparison. Cell viability was determined from triplicate culture wells by a CellTiter 96 Aqueous non-radioactive cell proliferation assay (Promega) at different time points and presented as mean and standard deviations.
Figure 4.
Induction of TRAIL receptors by N-terminal region in gelsolin fragments.
FACS analysis was performed to determine the expression levels of TRAIL-R1 (panel A) and TRAIL-R2 (panel B) on LX2 cell surface treated with CM from Huh 7 cells transfected with gelsolin fragments 1–70 (dark green line), 1–450 (orange line) and 450–782 (red line). Expression of these proteins in cells treated with media (blue line) was included as control. Cells were either treated with specific monoclonal antibody or isotype control antibody (light green line), followed by treatment with FITC-conjugated secondary antibody for FACS analysis.
Figure 5.
Activated but not quiescent LX2 cells undergo caspase 3 mediated apoptosis in presence of IHH CM.
Panel A: Comparative study of cell viability between quiescent and activated LX2 cells in the presence of IHH CM. Effect of normal medium (untreated) used for both quiescent and activated LX2 cells was included for comparison. Cell viability was determined from triplicate culture wells by a CellTiter 96 Aqueous non-radioactive cell proliferation assay (Promega) at different time points and presented as mean and standard deviations. Panel B: Expression of pro- and cleaved caspase 3 in quiescent and activated LX2 cells incubated with IHH CM. Panel C: LX2 cells incubated with CM with or without prior treatment with immobilized anti-gelsolin G1 antibody. Panel D: Expression of procaspase 9 in LX2 cells in the presence of IHH CM. Unused medium (SAGM) was included as a negative control in these experiments. Cellular actin was used as an internal control to verify the level of protein load in each lane.