Fig 1: Knockdown of CMTM6 enhances inflammation stimulated by hyperosmolarity in HCE-T cells. Knockdown of CMTM6 using shRNA promotes inflammation stimulated by hyperosmolarity in HCE-T cells. A lentivirus encoding a CMTM6 shRNA was used to silence CMTM6 expression. A lentivirus expressing control shRNA (shNC) was used as a negative control. (A) RT-PCR was used to detect the mRNA level of CMTM6 in HCE-T cells. (B) Western blot analysis was used to examine the protein level of CMTM6 in HCE-T cells. (C–E) Cultured cells were collected to assess the mRNA expression of TNFA (C), IL1B (D), and IL6 (E). (F) IL-6 release was quantified in the culture supernatant using CBA. Six groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 2: CMTM6 suppresses inflammation through the NF-κB/p65 pathway in HCE-T cells under hyperosmotic stress. (A, B) The effect of CMTM6 on the activation of p38 (A), Erk (A), and p65 (B) was examined using western blot analysis. (C–G) For the treatment of p65 inhibition, 50 µM JSH-23 was added to the cell culture medium, followed by incubation with 500-mOsM media for 8 or 24 hours. DMSO was used as a control. (C) The effect of JSH-23 was detected using western blot analysis. (D–F) The mRNA levels of TNFA (D), IL1B (E), and IL6 (F) were examined using RT-PCR. (G) The protein level of IL-6 in cell culture supernatants was detected using CBA. Six groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: 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.
Fig 4: CMTM6 attenuates inflammation stimulated by hyperosmolarity in HCE-T cells. A TG006-CMTM6-IRES-EGFP lentiviral vector was used to overexpress CMTM6. The TG006 lentiviral vector was used as a negative control. (A, B) Overexpression of CMTM6 in HCE-T cells was validated using RT-PCR (A) and western blot analyses (B). (C–E) RT-PCR was used to detect the mRNA levels of TNFA (C), IL1B (D), and IL6 (E). (F) The protein level of IL-6 in the culture supernatant was measured using CBA. Two-group statistical analysis was conducted using the unpaired Student's t-test (A). Four groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test (B–F). *P < 0.05, **P < 0.01, ***P < 0.001.
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