Fig. 3: POM121 alters expression of PPARγ target genes.

A POM121 KO reduces basal- and ligand-mediated protein expression of luciferase enzyme encoded on episomal reporter plasmids driven by DNA-binding motifs for PPARγ protein. HT29 clonal cells were transfected with a plasmid containing 3xPPREs from the enhancer region of the ACOX1 gene followed by incubation with vehicle (DMSO) or rosi (1–10 µM) for 48 h. Luciferase activity was normalized to protein content and expressed as -fold ± S.E. (*p < 0.05 vs. vehicle or EV, 2way-ANOVA with Bonferroni post-tests, n = 3 per clone). B POM121 KO decreases basal- and ligand-mediated protein expression of PPARγ target genes. Clonal cells were treated as in (A), followed by extraction as total cell lysate (TCL). Quantitative analyses (left) and representative images (right) from Western blots. O.D. values from gels normalized to HSP90 are -fold ± S.E. (*p < 0.05 vs. EV, 2way-ANOVA with Bonferroni post-tests, n = 3 per clone). C POM121 KO increases basal- or ligand-mediated mRNA expression of PPARγ target genes. Clonal cells were treated as in (A), followed by RNA extraction. Ct-values from RT-qPCRs normalized to B2M are -fold ± S.E. (*p < 0.05 vs. vehicle or EV, 2way-ANOVA with Bonferroni post-tests, n = 3 per clone). D POM121 KO and PPARγ agonist alter the subcellular distribution of PPARγ protein. Clonal cells were treated with vehicle (DMSO) or 10 µM rosi for 48 h and subjected to subcellular fractionation (SCF). Representative images (top right) and quantitative analyses (bottom) from Western blots. O.D. values of bands in gels normalized to HSP90 or lamin A/C are -fold ± S.E. (*p < 0.05 vs. vehicle or EV, 2way-ANOVA with Bonferroni post-tests, n = 3 per clone). Legend: CYT = soluble cytoplasm; NUC = soluble nucleoplasm; INS = insoluble fraction (cytoskeleton and membrane proteins).