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.