Fig. 5

NR5A2 promotes stemness via direct binding to the SOX2 promoter/enhancer. A qPCR analysis for mRNA levels of stemness-associated genes following 24 h of treatment with Cpd3 (40 µM, n = 4 biological replicates with four technical replicates). The dotted line indicates baseline expression levels, set as 1.0. B Time course for SOX2 RNA levels following Cpd3 treatment (40 µM) at 24, 48, and 72 h. C Immunofluorescence for SOX2 (yellow) following control (Ctrl) DMSO (top) or Cpd3 (bottom) treatment (40 µM) for 72 h. Nuclei are stained with DAPI (blue). D Western blot for SOX2 protein levels following NR5A2 overexpression (left) or 72 h of treatment with Cpd3 (40 µM) (right). E qPCR analysis of mRNA levels for NR5A2 and SOX2 in response to knockdown of NR5A2 using two different shNR5A2 (sh) in three different PDAC cultures. The dotted line indicates baseline expression levels, set as 1.0. F qPCR analysis of mRNA levels for NR5A2 and SOX2 in response to NR5A2 overexpression (OE) in two different PDAC cultures. G In vivo tumorigenicity of decreasing numbers of the most differentiated CD133^–FLUO^– pancreatic cancer cells following overexpression (OE) of NR5A2 or SOX2. H Percent input of immunoprecipitated DNA at CDKN1A positive control enhancer, NR5A2 and SOX2 promoters. The intergenic region is used as a negative ChIP control. I CUT&Tag analysis of NR5A2 protein binding at the SOX2 locus. WashU Epigenome browser tracks showing CUT&TAG signals at the SOX2 locus with the indicated transcription start site (TSS). Red signals represent NR5A2 binding in CD133⁺ PDAC cells (upper track), and purple signals represent NR5A2 binding in CD133^- PDAC cells (middle tracks). The black tracks represent the control, consisting of IgG binding in unsorted cells. In panels B, E, F, G, and I, data are presented as mean ± SD and statistically analyzed using two-tailed Mann–Whitney tests to compare two groups (n = 4 biological replicates). Asterisks indicate significance at the indicated levels: * p < 0.05 and **** p < 0.0001