Fig. 5: GP73-mediated secretion of PKM2 and GP73 promotes monocyte-to-macrophage differentiation.

A Representative IF staining of GP73, PKM2, and CD206 in high- and low- GP73 group of human HCC tissues. Scale bar, 100 μm. B The mRNA levels of M1 and M2 macrophage marker genes were measured using Real-time PCR after THP-1 cells incubation with indicated protein. C Flow cytometry was used for analysis MFI of M1 and M2 marker genes after THP-1 cells incubation with the indicated purified protein. D Schematic diagram of experimental design to establish the animal model. E Top, representative IF images of CD206 (red, M2 macrophage marker) and F4/80 (green, macrophage marker) expression in mouse livers educated with the indicated protein. Scale bar, 20 μm. Bottom, the statistical graph of double-positive cells per field. The arrows indicate double-positive cells. White insets depict a higher magnification of the arrows showing the representative area. F Top, representative images of Masson and Sirius red staining in the livers of mice in the indicated groups. Bottom, a statistical graph of the positive area of Masson and Sirius red staining in the livers of mice in the indicated groups. Scale bar, 100 μm. G Representative IHC images of α-SMA (top) and Collagen I (bottom) in the livers of mice in the indicated groups. Scale bar, 100 μm. H Content of ALT and AST from the blood of mice in the indicated groups. I Schematic diagram of experimental design to establish the cell co-culture model. J-L PKM2 knockdown increases M1 macrophage marker expression and decreases M2 macrophage marker expression. Real-time PCR (J), flow cytometry (K), and IF assay (L) were conducted with THP-1 cells after incubation with the indicated culture medium of the Huh7 cells. CD80 (red), CD163 (green), DAPI (blue), nucleus. Scale bar, 5 μm. M The protein levels of α-SMA, Collagen I, Collagen III, and Fibronectin in LX2 cells incubated with the indicated culture medium Of Huh7 cells were measured by western blotting. β-actin was used as a loading control. N Representative IF images of α-SMA and Fibronectin expression in LX2 cells incubated with the indicated CM of Huh7 cells. α-SMA (red), Fibronectin (green), DAPI (blue), nucleus. Scale bar, 10 μm. O Left, representative cell adherence assay images of Huh7 cells incubated with the indicated CM of Huh7 cells. Right, the quantitative statistic of OD 595 value. Scale bar, 200 μm. P Representative images of Transwell assay of Huh7 cells incubated with the indicated CM of Huh7 cells. Scale bar, 200 μm. Q The protein levels of E-cadherin, Vimentin, and N-cadherin in Huh7 cells incubated with the indicated CM were measured by western blotting. β-actin was used as a loading control. Error bars represent mean ± SD (n = 3). *p <0.05, ** p <0.01, *** p <0.001.