Fig. 4

HBeAg promoted the activation of hepatic stellate cells in a macrophage-dependent manner. LX-2 stellate cells were treated with HBeAg (2 μg/ml), CM-C, or CM-E from RAW264.7 macrophages (A) or U937 cells (B) for 24 h, the expression of α-SMA, collagen 1a1, and fibronectin was tested by qRT-PCR. Similarly, their expression in LX-2 cells and primary hepatic stellate cells was tested by western blot assay (C, D). LX-2 stellate cells were treated with HBeAg (2 μg/ml), CM-C, or CM-E from U937 cells for 24 h; the contractility, proliferation, and migration phenotypes were detected (E–G). Log2-transformed phosphorylation signal of enriched pathways following treatment with HBeAg or CM-E was detected. The signal was normalized to respective total protein and then to CM control. Heat map: compared to vehicle, red indicates increased signal, black no change, and green reduced signal (H). Key signaling intermediates for enriched pathways of CM-E were verified using western blots (I). LX-2 cells were treated with CM and inhibitors of enriched pathways (10μΜ) or the same volume of DMSO as control, then the contractility, proliferation, and migration phenotypes were tested (J–L). RAW264.7 macrophages were stimulated with HBeAg (2 μg/ml) at different time points, and the secretion of cytokines, chemokines, and growth factors was detected using the Magnetic Luminex Performance Assay (M). *P < 0.05, **P < 0.01, ***P < 0.001