Fig 2: NFAT3 protein binds CXCR4 promoter.HeyA8 cells were cultured as monolayer and as 3D spheroids (100,000 cells per spheroid) as described in materials and methods. (A) CXCR4 gene sequence according to GeneBank accession number AF005058. Regions with putative transcriptional factor binding consensus sequences are underlined and in bold. The block arrows indicate real-time PCR primers used for the chromatin immunoprecipitation analysis. (B) Chromatin immunoprecipitation of adherent HeyA8 cells and 3D spheroid showed significant (p <0.005) increase in relative abundance of NFAT3 and Pol II on the CXCR4 promoter. (C) Similar to 3D spheroids, adherent cells treated with 20 µM of U0126 showed significant (p <0.05) increases in relative abundance of NFAT3 and PolII on the CXCR4 promoter. Average values were derived from 2 independent IPs with qPCRs for each IP performed in duplicate (n = 4).

Fig 3: 3D spheroids enhance NFAT3 and CXCR4 expression.PCR array of transcription factors was performed according to manufacturer's protocol. The 3D spheroids have 100,000 cells per spheroid. (A) A list of transcriptional factors with the corresponding fold change in mRNA expression in cultured 3D spheroids relative to adherent HeyA8 cells. (B) NFAT3 mRNA showed an average of ∼ 20 fold increase in the 3D spheroid. (*, P <0.05 vs Adh, 1-way ANOVA). (C) Corresponding to the increase in NFAT3 in 3D spheroids, real-time PCR showed a 30-50-fold increase in CXCR4 mRNA in 3D spheroids when compared to adherent HeyA8 cells. (*, P <0.05 vs Adh, 1-way ANOVA). (D) Adherent and 3D spheroids western blot analysis of CXCR4 protein in HeyA8, MCF-7, and MDA-MB-231 showed higher CXCR4 levels with 3D spheroids than with adherent cells. (E) siRNA targeting NFAT3 decreased the number of CXCR4 positive cells compared to siCtr as determined by flow cytometry. Western blot analysis showed decreased NFAT3 following siNFAT3 vs siCtr and CXCR4 expression also decreased following siNFAT3. Flow cytometry of CXCR4 expression in 3D spheroids were pooled representative cells isolated from at least 10 individual spheroids. Real-time PCR results were normalized to actin. (*, P <0.05 vs siCtr, 1-way ANOVA). The PCR array results are the average of 2 separate experiments, the NFAT3 mRNA, CXCR4 mRNA, siNFAT3, and western blot experiments are representative of 3 separate experiments.

Fig 4: NFAT3 regulates CXCR4 expression.HeyA8 cells were cultured as a monolayer (adherent) and as 3D spheroids as described in materials and methods. HeyA8 adherent cells were treated with calcineurin-NFAT inhibitor cyclosporin A (1 µM) andFK506 (3 µM) for 72 hours. (A) Flow cytometry analysis of HeyA8 adherent cells showed basal CXCR4 surface protein expression was attenuated with the treatment of cyclosporin A as compared to no treatment group (Basal CXCR4). (***, P <0.0001 vs. CXCR4, 1-way ANOVA). (B) HeyA8 adherent cells treated with FK506 showed basal CXCR4 surface expression decreasing similar to cyclosporine A treated cells. (***, P <0.0001 vs. CXCR4, 1-way ANOVA). (C) HeyA8 3D spheroids were treated with Cyclosporin A (1 µM) for 7 days. Flow cytometry showed decreasing levels of surface CXCR4 protein expression treated with cyclosporin A. (**, P <0.05 vs. 3D spheroid, 1-way ANOVA). (D) Picture of HeyA8 3D spheroids from Fig. 6C. Cyclosporin A treated 3D spheroids compared to 3D spheroid control showed cells dissociated from the 3D spheroids and attaching to the bottom as adherent monolayer. (E)(F) Flow cytometry analysis of adherent HeyA8 and MCF-7 cells treated with ionomycin showed significant increase in Basal CXCR4 expression (**, P<0.05 vs. CXCR4, 1-way ANOVA) compared to no treatment (Basal CXCR4). Fig. 6 results are representative of 3 experiments.