Figure 3.

Spinning condition compared to static condition enhances TGF-β-induced epithelial-to-mesenchymal transition (EMT) pathway upregulation and inhibition by SB431542 treatment in bioprinted hepatic constructs. (A) Morphology of the surface and interior areas of bioprinted hepatic constructs in each group on day 10. Subgroups were divided into nontreated vehicle control, TGFβ-treated and combination of TGFβ with SB431542. Scale bar = 1000 μm. (B) Representative view of growth based on images for H&E staining of bioprinted hepatic constructs treated with TGF-β or TGF-β and SB431542 together under spinning and static conditions on day 10. Scale bar = 1000 μm. (C) qRT-PCR analysis of TWIST1, SNAIL1, SNAIL2, CDH1 and CDH2 genes that are involved in the EMT pathway in bioprinted hepatic constructs under static and spinning conditions on day 10. Error bars represent the means ± S.D. from three separate experiments. One-way ANOVA followed by Bonferroni’s test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 show significance between control and other group under indicated culture condition. # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 denote significance among each group under indicated culture condition. (D) Western blot analyses of total cell lysates from bioprinted hepatic constructs using anti-fibronectin, anti-phosphosmad2/3, anti-Smad2/3 and anti-β-actin antibodies. β-Actin served as a loading control. (E) Quantification of fibronectin expression normalized to β-actin and phoshpho-Smad2/3 expression normalized to Smad2/3. One-way ANOVA followed by Bonferroni’s test was used for statistical analysis. Error bars represent the means ± S.D. from three separate experiments. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicate significance between control and other group under indicated culture condition. # p < 0.05, ## p < 0.01 and #### p < 0.0001 show significance among each group under indicated culture condition.