Fig. 4: Impact of esophageal microenvironment on eosinophil phenotype.

A Experimental schema of eosinophil and EPC2 co-culture. Created in BioRender. Felton, J. (2025) https://BioRender.com/hhmye42. B Western blot showing JUN (top) and ATF3 (bottom) expression in CRISPR-transfected EOL-1 cell lines: wild-type EOL-1 (WT) and JUN knock-out (KO), ATF3 KO, and JUN/ATF3 double KO cells, representative of n = 3 experiments. C PCA plot showing bulk RNA sequencing of WT and ATF3-KO cells co-cultured with EPC2 cells for 3 days. D Gene ontology analysis of enriched gene pathways between WT and ATF3-KO co-cultured cells. E C3AR1 gene expression levels in WT, ATF3-KO and JUN-KO cells co-cultured with/without EPC2 cells for 3 days in vitro (WT and ATF3-KO: n = 10, JUN-KO: n = 12, 4 independent experiments). Scatter plots show the mean with standard deviation error bars. Statistical analysis shows two-way ANOVA with Tukey’s multiple comparisons. F Bar chart, with representative histograms showing expression of C3ar1 on WT and transcription factor KO EOL-1 cells co-cultured with EPC2 cells for 3 days in vitro (WT: n = 7, ATF3-KO: n = 8, JUN-KO: n = 9, 3 independent experiments). Scatter plots show the mean with standard deviation error bars. Statistical analysis shows two-way ANOVA with Tukey’s multiple comparisons. For (E) and (F), WT EOL-1 cells (white). WT cells co-cultured with EPC2 cells (gray). ATF3-KO cells (light blue). ATF3-KO cells co-cultured with EPC2 cells (dark blue). JUN-KO cells (light orange). JUN-KO cells co-cultured with EPC2 cells (dark orange).