Fig. 5.

LYZ promotes HCC cell proliferation and migration in the paracrine manner. (A) The indicated cells were cultured in E-Plate 16 wells and treated with or without recombinant LYZ (1 mg/mL), together with LYZ-specific antibody (10 μg/mL), TAG (0.1 mg/mL), or control IgG (10 μg/mL). Cell proliferation was monitored by RTCA system for the indicated time periods. (B) Huh-7 or Hep3B cells were cultured in the HepG2 supernatant supplied with LYZ-specific antibody (10 μg/mL), TAG (0.1 mg/mL), or control IgG (10 μg/mL). Cell proliferation was monitored by RTCA. (C) The indicated cells were cultured in either control (shCtrl) or LYZ knockdown (sh2/sh3) HepG2 supernatant, supplied with or without recombinant LYZ (1 mg/mL). Cell proliferation was monitored by RTCA. (D) Transwell assay to examine the effect of LYZ on Huh-7 cell migration. Huh-7 cells were plated in the Boyden chamber inserts with or without treatment of recombinant LYZ (1 mg/mL) in the lower chambers, together with the LYZ-specific antibody (10 μg/mL), TAG (0.1 mg/mL), or control IgG (10 μg/mL). Representative fields of migrated cells and summarized data are shown. (E–G) Huh-7-Luc cells together with either WT (N = 7) or LYZ KO (N = 7) HepG2 cells were coinoculated into NOD/SCID mice as described in Materials and Methods. The in vivo growth of Huh-7-Luc cells was determined by IVIS. (E) Scheme of the coinoculation experiment. (F) Summarized results for the average radiance of luciferase activity in mice treated as indicated. (G) Images of the radiance of luciferase activity in each mouse treated as indicated. Data in A–D and F are shown as the mean ± SEM and are representative of at least three independent experiments. [Scale bars in D represent 200 μm (Upper) or 100 μm (Lower).] **P < 0.01, ***P < 0.001 (two-way ANOVA followed by Tukey’s multiple comparisons for A–C and F; one-way ANOVA followed by Tukey’s multiple comparisons for D).