Fig 2: Cell proliferation and the cell cycle depending on TCF4 expression. (A) Cell proliferation rate by TCF4 inhibition (si-TCF4) or TCF4 overexpression (pl-TCF4). N = 7, P = 0.026, and 0.038. (B–D) Cell cycle analysis showed the percentage of cells in the S-phase. (E–H) The expression of phospho-extracellular signal–regulated kinase 1/2 (pERK1/2) was shown. (I–L) Cyclin-dependent kinase 1 (CDK1) expression is shown. N = 4, P = 0.008 and 0.007. (M–P) Cyclin D1 expression. (Q–T) CDKN2A expression. All the measurements were conducted in triplicate or quadruplicate. ***P < 0.001, **P < 0.01, and *P < 0.05 statistically significant by independent t-test.

Fig 3: Effect of TCF4 CRISPR activation on corneal endothelial wound healing. (A, B) Corneal opacity in control, a-TCF4 group, and si-TCF4 group. (C) TCF4 expression (green) was increased in the cornea endothelium of the a-TCF4 group. Nuclear staining was performed with Hoechst 33342 (blue). (D) qRT-PCR showed the TCF4 expression levels. (E) ß-catenin expression in the corneal endothelium. (F, G) Corneal endothelium stained with alizarin S red. Alizarin S red staining showed the increased wound healing in the corneal endothelium in the a-TCF4 group. The number of corneal endothelial cells was higher in the a-TCF4 group. (H) MitoTracker green fluorescence indicates mitochondrial mass and mitoSOX red fluorescence indicates oxidative stress levels. All the measurements were conducted in triplicate or quadruplicate. ***P < 0.001, **P < 0.01, and *P < 0.05 by independent t-test.

Fig 4: Culture and identification of hCECs and Wnt signaling regulated by TCF4. (A) The hCECs were cultured in mosaic pattern. (B, C) TCF4 expression after treating siRNA for TCF4 (si-TCF4) and CRISPR/dCas9 activation system for TCF4 (pl-TCF4). (D–G) ß-catenin expression was not different. (H–K) Activation of GSK3ß was not different. (L–O) GFAP expression was regulated by TCF4. All the measurements were conducted in triplicate or quadruplicate. ***P < 0.001, **P < 0.01, and *P < 0.05 statistically significant by independent t-test.

Fig 5: Role of TCF4 in mitochondrial functions. (A–C) Mitochondrial viability stain showed that mitochondrial viability was regulated by TCF4. N = 7, P = 0.008, and 0.037. (D–F) Mitochondrial mass was evaluated by Mitotracker Green fluorescence. Mitochondrial mass was regulated by TCF4. Bar scale = 50 µm. (G–I) Mitochondrial superoxide production was evaluated by MitoSOX Red fluorescence. Mitochondrial superoxide formation was regulated by TCF4. Bar scale = 50 µm. (J) Co-localization of MitoTracker green and MitoSOX. (K–M) Intracellular oxidative stress levels were evaluated by DCF fluorescence (green). Bar scale = 50 µm. N = 6, P = 0.026, and 0.001. (N, O) Relative adenosine triphosphate (ATP) production was evaluated. (P–S) Activation of AMP-activated protein kinase (AMPK) was evaluated. All the measurements were conducted in triplicate or quadruplicate. ***P < 0.001, **P < 0.01, and *P < 0.05 statistically significant by independent t-test.