Journal: The Journal of Biological Chemistry

Article Title: RhoE downregulation leads to enhanced cholesterol biosynthesis and sorafenib resistance in hepatocellular carcinoma

doi: 10.1016/j.jbc.2025.110918

Figure Lengend Snippet: Activation of the cholesterol synthesis pathway in HCC cells mediates drug resistance after sorafenib treatment . A–C , protein levels of HMGCR, p-MEK, MEK, p-ERK, and ERK were analyzed by Western blotting following sorafenib treatment in HepG2, Huh7, and MHCC97H cells. D , Western blot analysis of HMGCR protein expression in parental HepG2 cells and two sorafenib-resistant HepG2 cell lines. E–G , following 24-h cotreatment with sorafenib and the FAK inhibitors defactinib and PF-573228, HMGCR, p-FAK, and FAK protein levels were analyzed by Western blotting in HepG2, Huh7, and MHCC97H cells. H , FAK, p-FAK, and HMGCR protein levels were examined by Western blotting in WT and FAK-KO Huh7 cells following 24-h sorafenib treatment. I , either WT FAK or the Y397F mutant was reintroduced into FAK-KO Huh7 cells, followed by 24-h sorafenib treatment. HMGCR, FAK, and p-FAK protein levels were analyzed by Western blotting. J , Huh7 cells overexpressing HMGCR were cotreated with defactinib and sorafenib for 48 h, and cell viability was assessed using the Alamar Blue assay. K , cellular cholesterol levels in Huh7 cells were measured using a cholesterol assay kit under basal conditions. Data are presented as mean ± SD (n = 3). L , cholesterol levels in parental HepG2 and two sorafenib-resistant HepG2 cell lines were measured using a cholesterol assay kit. M , cellular cholesterol levels in Huh7 cells were measured using a cholesterol assay kit following 24-h co-treatment with defactinib and sorafenib. N and O , cell viability in Huh7 cells was measured using the Alamar Blue assay following 72-h cotreatment with simvastatin or lovastatin in combination with sorafenib. P , cell viability in Huh7 cells was measured using the Alamar Blue assay following 72-h cotreatment with exogenous cholesterol and the combination of defactinib and sorafenib. Statistical significance for panel K was determined using unpaired two-tailed Student’s t test. Statistical significance for panels J and L–P was determined using two-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD from at least three independent experiments. Significance is indicated as p ≥ 0.05 (ns), p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), and p < 0.0001 (∗∗∗∗). FAK, focal adhesion kinase; p-FAK, phosphorylated FAK; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Article Snippet: Defactinib (HY-12289), PF-573228 (HY-10461), sorafenib (HY-10201), lovastatin (HY-N0504), and simvastatin (HY-17502) were obtained from MedChemExpress.

Techniques: Activation Assay, Western Blot, Expressing, Mutagenesis, Alamar Blue Assay, Cholesterol Assay, Two Tailed Test