Fig 2: CMTM6 reduces barrier disruption in vivo and in vitro. (A) Immunofluorescence analysis of ZO-1 in wholemount corneal tissues with quantification by ImageJ. Scale bars: 20 µm. (B–E) Changes in the barrier resistance of HCE-T cells were examined using ECIS. (B) Resistance measurements generated at 4000 Hz from HCE-T cells grown in 310- and 500-mOsM culture media. (C) The endpoint resistance values of HCE-T cells. (D) Rb parameters were measured to reflect the paracellular barrier strength after the stabilization of resistance. (E) The endpoint Rb values of HCE-T cells. (F, G) After Cmtm6 knockdown in HCE-T cells using siRNA (F) or shRNA (G), protein levels of ZO-1 were detected using western blot analysis. Results are shown as mean ± SD. Four groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test (C, E–G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: CMTM6 is expressed at a high level in corneal and conjunctival epithelium. (A) The tissue and organ distributions of Cmtm6 were analyzed within the BioGPS database. (B) The detection of CMTM6 expression in adult human corneas and adjacent conjunctivas was carried out utilizing GSE182583 (n = 4). (C) The expression of Cmtm6 in corneal epithelium, corneal stroma, and conjunctiva of healthy mice was assessed by RT-PCR (n = 5 in each group). (D, E) The protein level of CMTM6 was evaluated by IHC. Representative images of cornea (D) and conjunctiva (E) are shown. Scale bars: 20 µm. The results of a one-way ANOVA and Dunnett's multiple comparisons test are displayed as mean ± SD; “n” represents both eyes from a single mouse (C). **P < 0.01, ****P < 0.0001. CS_Epi, corneal superficial epithelial cells; CB_Epi, corneal basal epithelial cells; CSKs, corneal stroma keratocytes; C_Endo, corneal endothelial cells; FC_Endo, fibroblastic corneal endothelial cells; Conj_Epi, conjunctival epithelial cells.

Fig 4: CMTM6 alleviates damage in DE mice. (A, D) CFS was used to evaluate corneal epithelial defects. (B, E) Tear production was examined using the phenol red thread test. (C, F) The conjunctival goblet cells were stained with PAS, and the positive cells were counted. Scale bars: 20 µm. Results are shown as mean ± SD (n = 6–8 in each group). A two-group statistical analysis was conducted using the unpaired Student's t-test; “n” represents one eye per mouse (A–F). **P < 0.01, ***P < 0.001; ns, non-significant.

Fig 5: CMTM6 reduces DLNs Th1 and Th17 cell infiltration and proinflammatory cytokine production in DE mice. (A) Representative flow cytometry plots and quantitative summary of Th1, Th17, and Treg percentages among the DLNs. (B–D) RT-PCR was used to examine the production of proinflammatory cytokines Tnfa (B), Il1b (C), and Il6 (D) in corneas. (E) The expression of TNF-α in tears was measured using CBA. Results are shown as mean ± SD (n = 3–4 in each group). A two-group statistical analysis was conducted using the unpaired Student's t-test; “n” represents bilateral draining lymph nodes (A) or both eyes (B–E) from a single mouse. *P < 0.05, **P < 0.01, ***P < 0.001.