Fig. 4

NFs/CAFs with modulated EREG expression regulate the migration and invasion of oral squamous cell carcinoma cell lines. a, CCK8 assay showing that CAF CM significantly promoted HSC3 cell viability compared with NF CM. However, alteration of EREG expression in the same type of fibroblasts did not significantly affect their influence on HSC3 cell viability. b, Quantification of colony formation revealed that CAF-siNC CM significantly promoted HSC3 proliferation compared with NF-pcDNA3.1 CM. However, alteration of EREG expression in the same type of fibroblasts did not significantly affect their influence on HSC3 proliferation. c, Representative images and transwell migration and invasion assays with the HSC3 cell line. EREG upregulation induced migration/invasion-promoting CAF-like functions in NFs, but this effect was attenuated by AG490 treatment. On the other hand, EREG downregulation in CAFs interfered with their migration/invasion-promoting ability but was restored by rIL-6 treatment. d, Representative HE staining images of a 3D invasion model using tissue engineering. The results show that CAFs significantly promoted HSC3 invasion compared with NFs, but their invasion-supportive ability was attenuated after EREG knockdown. Treatment with rIL-6 restored the invasion-promoting abilities of EREG-low CAFs. On the other hand, EREG overexpression in NFs led to an increased invasion-promoting ability, but this effect was attenuated by AG490 treatment. e-g, Quantification of transwell assays and 3D invasion assays. H&I, Representative images and quantification of EMT marker (E-cadherin, N-cadherin, vimentin, SMA, and Snail) expression in HSC3 cells after the indicated treatment. GAPDH was used as a loading control. *: p < 0.05, **: p < 0.01