Fig. 2: SOX4 Complexes with STAT6 in HCC Cells to Regulate IL-4-Mediated STAT6 Phosphorylation.

A Immunoprecipitation (IP) assays were performed using 1 mg of Hep3B or SNU-475 cell lysates incubated with SOX4-specific or IgG control antibodies. Immunoprecipitated proteins were analyzed by western blotting for STAT6 and phosphorylated STAT6 (p-STAT6-Y641), with input lysates (1/20 volume) and actin serving as controls. B HEK293 cells were co-transfected with full length CBP-tagged SOX4 and STAT6 constructs, including various STAT6 deletion mutants. Co-immunoprecipitation was performed using CBP beads, followed by detection of the Flag-tagged STAT6 constructs with M2 (Flag) antibodies. Input protein (1/20 volume) expression was validated via western blotting. C Flag-tagged SOX4 deletion constructs were co-transfected with full-length STAT6 expression plasmids in HEK293 cells. SOX4 pull-downs were probed for STAT6, and western blotting confirmed input protein expression. D, E Immunofluorescence staining of fixed Hep3B cells (D) and HCC tumoroids (E) demonstrated colocalization of SOX4 and STAT6 in the nucleus. Secondary antibodies were conjugated with distinct colors, and localization was visualized by confocal microscopy. F Hep3B, Hep3B SOX4^−/−, and SNU-475 cells transfected with scramble or SOX4 si were treated with IL-4. Western blot analysis was performed to assess p-STAT6-Y641, STAT6, and SOX4 expression, with actin as a loading control. After normalization with actin, IL-4-induced phosphorylation of p-STAT6-Y641 was quantified as p-STAT6/STAT6 (right panel), the ratio of p-STAT6-Y641/STAT6 in Hep3B or SNU475 cells treated with IL-4 was set as the baseline (1-fold). The corresponding protein expression in SOX4^−/− or SNU-475 transfected with SOX si cells was normalized to the expression level in Hep3B or SNU475 cells treated with IL-4 and presented as a relative fold change. G RANKL, an IL-4-STAT6 downstream gene, was examined by qRT-PCR by its specific primers in Hep3B and Hep3B SOX4^−/− cells treated with or without IL-4. 18S served as an internal control for gene expression normalization. The expression levels of RANKL in Hep3B and SOX4^−/− cells, with or without IL-4 treatment, were calculated using the ΔCt method. The Ct value of RANKL was normalized to that of the housekeeping gene 18S (ΔCt = Ct_RANKL - Ct_18S). The expression level of RANKL in untreated Hep3B cells was set as the reference (One-fold). RANKL expressions in other conditions were then normalized to this reference and presented as relative fold change. Western blot and qRT-PCR results were obtained from three independent experiments, with data presented as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).