Fig. 4. DEX treatment enhances ROR signaling (ROR1, ROR2, and Wnt5a) and stemness phenotype in OC spheroids while promoting cell differentiation.

a Western blot analysis of OVCAR3/OVCAR3cis cells grown in spheroid conditions in the presence or absence of 100 nM DEX treatment for 5 days. β-tubulin was used as a loading control. A marked increase in ROR signaling (Wnt5a, ROR1, and ROR2 levels) and stemness markers (BMI-1, ALDH1A1, and YAP/TAZ) is observed in DEX-treated spheroids. b Photomicrographs of OVCAR3/OVCAR3cis grown in spheroid conditions and treated as in a, scale bar 400 μM. c Western blot analysis and quantification of E-cadherin and ZO-1 levels of OVCAR3/OVCAR3cis grown in spheroid conditions and treated as in a. β-tubulin was used as a loading control. Protein levels were normalized to β-tubulin and an untreated sample was used as a reference point (value 1) for quantification. d Hierarchical clustering of expression of mesenchymal, de-differentiated, cell type marker genes²³ shows that FMOC09 exhibits a mesenchymal-like expression pattern; high expression of SOX11, and low expression of kallikreins. Values are presented as log2 transformed transcripts per kilobase million (TPM) from RNA-Seq from five PDCs, which have been row-normalized (zero to one). e Western blot analysis of PDC spheroids in the presence or absence of 100 nM DEX treatment as indicated showing upregulation of ROR1, ROR2, and its downstream YAP/TAZ and BMI-1 markers, as well as ALDH1A1 and pAKT levels in DEX-treated samples. β-tubulin was used as a loading control. f Photomicrographs of PDCs grown in spheroid conditions and treated as in e, scale bar 100 μM. g–h Western blot analysis (g) and protein quantification (h) of differentiation markers in cell lysates derived from PDCs spheroids grown as in f. Protein levels were normalized to β-tubulin and an untreated sample was used as a reference point (value 1) for quantification. A representative of three technical replicates is shown for each panel.