CECs on the soft substrate showed a lower total YAP expression but higher cytoplasmic localization compared with those plated on the stiff substrate and dish (
Fig. 6C). This was consistent with an ex vivo pattern (
Fig. 3G). To further verify that YAP is involved in stiff-induced cell behavior changes in CECs, in a subsequent experiment, the small-molecule YAP inhibitor verteporfin (VP) was added to the stiff substrate at day 6. We found that VP treatment remarkably decreased YAP, TAZ, and TEAD1 expression in a dose-dependent manner (
Figs. 7A,
7G). We also found fibrotic proteins markers (vimentin, α-SMA, fibronectin, type I collagens) (
Figs. 7B,
7I) related to EnMT were downregulated in a dose-dependent manner. Immunostaining revealed a lower expression and cytoplasmic localization of YAP of CECs on the stiff substrate with 5 µM VP compared with those without any treatment (
Fig. 7C). Immunostaining also showed that through 5 µM VP treatment, the EnMT-related marker vimentin of CECs on the stiff matrix could be restored to its expression state on the soft substrate (
Fig. 7D). Importantly, although it had no effect on central CECs (
Supplementary Fig. S2), the stemness markers (Sox2, Oct3/4, and Nanog) (
Figs. 7E,
7H) of peripheral CECs were also upregulated by YAP inhibitor. Of note, we also found that the main content of DM and differentiation marker of CECs (
Figs. 7F,
7I), which were elevated on the stiff substrate, were also downregulated by YAP inhibitor in a dose-dependent manner. Therefore, it could be inferred that the substrate stiffness modulates the stemness maintenance, differentiation, and EnMT of CECs through the paxillin–YAP pathway.