VICs switch to a less activated and less proliferative fibroblast phenotype on softer substrates.

<p>(A) 6 hours after switching stiff gels to soft on day 3, VICs were collected for mRNA quantification based on real-time PCR. Myofibroblast gene markers, α-SMA and connective tissue growth factor (CTGF), were significantly down-regulated in soft and stiff-to-soft conditions, compared with stiff. The fibroblast gene marker, vimentin, was expressed at a similar level on different substrates. (B) To measure proliferation, VICs cultured on stiff, soft or stiff-to-soft gels were chased with EdU for 3 hours on day 5. EdU incorporation into DNA was detected by labeling EdU with Alexa Fluor 488 and was quantified via flow cytometry. Relative proliferation on day 5 of VIC culture was calculated based on normalizing the percent of EdU+ cells in each condition to that of the stiff condition. VICs were less proliferative on soft and stiff-to-soft gels than on stiff gels. (C) Representative EdU staining of VICs cultured on PD-PEG gels. As expected, EdU staining (green) is co-localized with nuclei (blue). Scale bar: 100 µm. * indicates p<0.05. (D) VICs cultured on plastic plate, stiff, soft or stiff-to-soft gels were fixed on day 5 and stained for vimentin. De-activated fibroblasts on stiff-to-soft gels maintain the mesenchymal fibroblast fate. Green: vimentin. Blue: nuclei. Scale bar: 100 µm.