Fig. 4: Enhanced expression of SERPINE1 (PAI-1) is associated with dysregulated cholesterol-TGF-β signaling and increased chromatin accessibility.

A Fold change of TGF-β1 protein in cell culture supernatant of HCT116 LD, MD and HD relative to PAR determined using ELISA. B Immunoblotting of total SMAD2/3 (tSMAD2/3), phospho-SMAD2 (pSMAD2), phospho-SMAD3 (pSMAD3) and SMAD4 in oxaliplatin-resistant HCT116 models. C, ATAC-seq signal tracks near the SERPINE1 gene. Red dotted box indicates area of differential peaks near 5′ UTR quantified for mean signal intensity. C’ Normalized mean signal intensity denoted as chromatin accessibility fold change in SERPINE1 promoter regions across oxaliplatin-resistant HCT116 models relative to PAR. D Expression fold change of cholesterol-related genes identified in Fig. 2A in the resistant models relative to parental cells determined using quantitative real-time PCR. E Fold change of total intracellular cholesterol in resistant models relative to parental cells determined using ELISA. F Representative immunoblots of TGFBRII and caveolin after fractionation. # indicates TGFBRII found in lipid raft (caveolin-1 [Cav1] high/positive) fractions. F’ Quantification of three independent immunoblots denoting the ratio of TGFBRII-positive fractions to Cav1 positive fractions. G Immunofluorescence staining for colocalization of cholera toxin subunit B (CTxB; green, lipid rafts) and TGF-β receptor II (TGF-βRII; red). White box denotes selected regions that are magnified. Red dotted box highlights the high oxaliplatin dose (HCT116 HD) that show most drug-tolerant persistent cells with reduced lipid rafts colocalized with TGF-βRII. Statistical significance was determined using Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test for (A, C’, E), and Ordinary 2-way ANOVA followed by Dunnett’s multiple comparisons test for D.