Skip to main content
. 2023 Jul 10;120(29):e2215744120. doi: 10.1073/pnas.2215744120

Fig. 3.

Fig. 3.

Neutralization of LYZ suppresses HCC cell proliferation and migration. (A) HepG2 or PLC/PRF/5 cells were cultured in the E-Plate 16 wells and treated with the indicated concentrations of LYZ-specific antibody (Ab5, 5 μg/mL; Ab10, 10 μg/mL) or TAG (0.1/0.5/1 mg/mL), or the isotype control IgG. Cell proliferation was monitored by RTCA system for the indicated time periods. Data are representative of three independent experiments. (B and C) Transwell assay for HepG2 (B) or Huh-7 (C) cells treated with the indicated concentrations of LYZ-specific antibody or control IgG. Representative fields of migrated cells and summarized data are shown for at least three independent experiments. (D–G) WT (N = 10) or LYZ KO (N = 7) HepG2 cells were inoculated subcutaneously into NOD/SCID mice as described in Materials and Methods. (D) Average tumor growth was measured by tumor size. (E and F) The image and weight of tumors from each group are shown. Each dot represents an individual tumor from each group. (G) IHC staining of Ki-67 expression in WT or LYZ KO tumors. Representative images and summarized data for Ki-67 staining scores are shown. Each dot represents the average Ki-67 staining score for at least three random fields of an individual tumor. (H–J) Huh-7-Luc cells were orthotopically inoculated into NOD/SCID mice as described in Materials and Methods, followed by intraperitoneal treatment with LYZ-specific antibody (200 μg/mouse, N = 10), Sorafenib (15 mg/kg, N = 8), or control isotype IgG (200 μg/mouse, N = 10) daily. (H) Scheme of the imaging and therapy schedule. (I) Average radiance of luciferase activity of Huh-7-Luc cells in mice treated as indicated. (J) IHC staining of Ki-67 expression in orthotopic tumors from mice treated as indicated. The results shown here are representative of at least three mice. Data in A–D and I are shown as the mean ± SEM. [Scale bars in B and C represent 200 μm (Upper) or 100 μm (Lower). Scale bars: G (50 μm); J (20 μm).] *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA followed by Tukey’s multiple comparisons for A and I, and by Sidak’s multiple comparisons for D; one-way ANOVA followed by Tukey’s multiple comparisons for B and C; two-tailed unpaired Student’s t test for F and G).