Fig. 6

HERC2 regulated JAK2/STAT3 signaling through direct interaction with PTP1B. A Huh7 cells were treated with 50 ng/ml IL-6 for 20 min. The extracted protein was precipitated with an antibody against HERC2 and separated by SDS-PAGE followed by silver staining. On the other hand, the band was analyzed with 4-D label-free quantitative proteomics. B Huh7 cells were treated with 50 ng/ml IL-6 for 20 min, and the interaction between HERC2 and PTP1B was validated by an immunoprecipitation assay. C-D HEK293T cells were co-transfected with HERC2-Flag and PTP1B-HA (C) or PTP1B-mcherry (D) plasmids. C Immunoprecipitation was performed using an anti-FLAG magnetic beads. The presence of coprecipitated HERC2 was determined by immunoblotting with the anti-HA antibody. D The colocalization of HERC2 and PTP1B in the cells was analyzed by immunofluorescence assay. E HERC2 and PTP1B double-deficient Huh7 cell lines were established. The cells were treated with 50 ng/ml IL-6 for 20 min, and the phosphorylation levels of JAK2 and STAT3 were detected by western blot analysis. F Cells were treated with 50 ng/ml IL-6 for 24 h. The RT-qPCR assay was used to detect the mRNA expression of CSC-related genes. G Cells were cultured with medium containing 100 × N2, 50 × B27, 20 ng/ml EGF, 10 nmol FGF, 5 μg/ml insulin, and 0.4% BSA for 7 days, scale bars = 100 μm. H The cells were treated with 20 μM sorafenib for 24 h. A flow cytometry assay was used to determine the percentage of apoptotic cells. I Cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. J and K Activated PBMCs were cocultured with HCC cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay (J). K IFN-γ levels of CD8⁺ T cells were determined by flow cytometry analysis. NS: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Data from one representative experiment of three independent experiments are presented