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

Fig. 6.

Fig. 6.

LYZ promotes HCC cell proliferation through interacting with csGRP78. (A) MS identification of LYZ-interacting cell surface proteins. Control vector (Vec) or Flag-LYZ were transfected into SNU-475 cells, followed by separation of PM fractions and immunoprecipitation with anti-Flag agarose beads. The immunoprecipitates were separated by SDS-PAGE and Coomassie blue staining, followed by MS identification of specific bands. (B and C) IP-WB analysis of the LYZ-GRP78 interaction. (B) Flag-LYZ and HA-GRP78 were cotransfected into HEK293T cells, followed by IP-WB analysis. (C) IP with PM fractions from SNU-475 cells as described in Materials and Methods, followed by WB analysis of LYZ-csGRP78 interaction. GRP78 and ATP1A1 from PM fractions of SNU-475 cells served as the IP control. (D) Confocal analysis of the colocalization of LYZ with GRP78 on the cell surface. PLC/PRF/5 cells were treated with or without monensin, followed by analysis of the colocalization of secreted LYZ with csGRP78. Representative confocal images are shown, with arrows indicating the colocalization of LYZ with GRP78 on the cell surface. (E and F) IP-WB analysis of the effects of TAG or LYZ mutates on LYZ-csGRP78 interaction. (E) IP with PM fractions from SNU-475 cells in the absence or presence of increasing concentrations of TAG (0.1-1 mg/mL), followed by WB analysis of LYZ-csGRP78 interaction. (F) Control vector, Flag-LYZ WT, or the indicated mutations were transfected into HEK293T cells, followed by treatment as described in Materials and Methods and IP-WB analysis of LYZmut-csGRP78 interaction. (G) Huh-7 cells were cultured in E-Plate 16 wells and treated with or without recombinant LYZ (1 mg/mL), together with anti-LYZ (10 μg/mL), anti-GRP78 (10 μg/mL), or control IgG (10 μg/mL) antibodies. Cell proliferation was monitored by RTCA. (H and I) SNU-475 cells were transfected with control or GRP78-specific siRNAs, followed by treatment with or without recombinant LYZ (1 mg/mL). Cell proliferation and migration were determined by RTCA and Transwell, respectively. (J and K) WB analysis of the indicated signaling pathways activation in differential SNU-475 cells stimulated with recombinant LYZ (1 mg/mL). Data in G and H are shown as the mean ± SEM and are representative of three independent experiments. (Scale bars in D and I represent 20 μm and 100 μm, respectively.) ***P < 0.001 (two-way ANOVA followed by Tukey’s multiple comparisons for G to I).