Fig 2: IHC staining of cytokeratin and AQP3 expression in wounds. a IHC images of wound sections stained with KRT1 on day 13. Scale bar = 20 µm. b Quantification of KRT1 IHC stained tissues. c IHC images of wound sections stained with AQP3 on day 13. Scale bar = 20 µm. d Quantification of AQP3 IHC stained tissues. In b and d, data are shown as mean ± SEM; n = 6 for each group. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001 vs vehicle control group

Fig 3: Medium supplementation on rhConE does not induce goblet cell differentiation. Media test of Pro3 vs. Pro10 on rhConE. (A) Histological analysis of in Pro3 or Pro10 cultured rhConE via H&E (left two columns) and AB/P (right two columns) after 10 days, 15 days, and 20 days of culture. (B) Immunofluorescence of CK1, CK13, CK14, and CK19 performed on rhConE cultured in Pro10 after 15 days. Blue = DAPI, green = respective cytokeratin. Scale bar = 100 µm. (C) MTT-assay of Pro3 vs. Pro10 cultured rhConE measured via optical density of 570 nm extinction (n = 3). (D) TEER1000 Hz measurement of rhConE cultured in Pro3 or in Pro10 medium over the culture time from 6 days until 20 days (n = 3).

Fig 4: PPP2R2A is epistatic to IER5.(A) Western blot showing IER5 and B55a protein levels in single (KO) and double (DKO) PPP2R2A and IER5 knockout clones in the presence of GSI (-) and 4 hr after GSI washout (+). (B) PPP2R2A knockout enhances Notch-dependent expression of KRT1. RT-PCR analysis of KRT1 expression in SC2 cells transduced with an empty retrovirus (SC2/EV); PPP2R2A knockout cells (B55 KO) transduced with empty retrovirus (B55KO/EV); and PPP2R2A knockout cells transduced with B55a-expressing retrovirus (B55KO/B55AB). WO = GSI washout. *, p<0.05; **, p<0.005 (two-tailed student t test). (C) Immunohistochemical (IHC) staining for involucrin of SC2 control, B55KO, and B55KO/B55AB cells in raft cultures in GSI-free medium. (D) B55a knockout negates the requirement for IER5 for Notch-dependent upregulation of KRT1. Results are shown for SC2 control cells (SC2/EV); an IER5 knockout clone; a PPP2R2A knockout clone (B55KO); and three IER5/PPP2R2A double knockout (DKO) clones. Cells were maintained in GSI or harvested 72 hr following GSI washout (WO). KRT1 transcript abundance was measured in biological replicates prepared in triplicate by RT-PCR and normalized against GAPDH. Error bars represent standard errors of the mean. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005 (all two-tailed student t test).

Fig 5: Full thickness conjunctival models (FTConM) displayed in vivo marker expression. Immunofluorescence staining of CK1, CK13, CK14, CK19, Vimentin, and E-Cadherin performed on FTConM after 10 days, 15 days, and 20 days of culture. Blue = DAPI, green = respective Cytokeratin/Vimentin, red = E-Cadherin. Scale bar = 100 µm.