Fig. 6: Expression of SOX4, STAT6, p-STAT6, and MTHFD2 in HCC Specimens and Their Impact on Metabolomics and Drug Resistance.

A Western blot analysis of SOX4, p-STAT6 (Y641), STAT6, and MTHFD2 in paired normal (N) and tumor (T) tissues from 62 HCC patients. Representative data from 12 patients are shown; results for 50 additional samples are in Supplementart Fig. 3. β-actin was used as a loading control. Patients receiving TKIs, immunotherapy, or combination treatment are highlighted in red. B Quantification of protein band intensities using ImageJ software (NIH, Bethesda, MD, USA), normalized to β-actin. Statistical significance: *P < 0.05, **P < 0.01. C Correlation analysis of protein expression levels. D Metabolomic analysis of SOX4/STAT6/MTHFD2^high and SOX4/STAT6/MTHFD2^low tumor samples (n = 21) using LC-MS. Differential metabolite abundance was analyzed with MetaboAnalyst 6.0 website (https://www.metaboanalyst.ca/home.xhtml) [59]. The heatmap (left) shows log-ratio values, with red indicating upregulation and blue indicating downregulation. Identified metabolites are numbered 1-58 (right). The abundance of metabolites was showed in log₁₀ value as scale bar. The sample number of clinical specimen was the same as the sample number present in Fig. 6A and Supplementary Fig. 3. E Clinical response and SOX4, STAT6, and MTHFD2 expression in 18 HCC specimens. Expression was normalized to β-actin, then further normalized as tumor/normal fold change. Colors indicate relative expressions (green to red). F Patient #6 (61 y/o male): Treated with atezolizumab and bevacizumab for 9 cycles, achieving near-complete response at 6 months, with treatment cessation at 8 months. Patient #8 (45 y/o male): Progressed despite sorafenib and transarterial chemoembolization.