Fig 1: GZMA modulated CREB nuclear translocation to promote intestinal epithelial integirty (A) HT-29 and Caco-2 cells were serum-starved for 24 h, followed by stimulation with GZMA (500 nM) for indicated time. Cell cytoplasmic and nuclear proteins were extracted to detect CREB. Data represent the mean ± s.d. of three independent experiments and were analyzed by one-way ANOVA with multiple comparisons, followed by Dunnett post hoc test for significance versus Control, ****p < 0.0001. the control was normalized as 1. (B) Immunofluorescence of CREB localization in HT-29 and Caco-2 cells treated with or without GZMA for 1 h after serum starved for 24 h. (C) western blotting was conducted to analyze the expression of the specific proteins in Caco-2 cells after transferd with sh-CREB plasmid, followed by stimulation with GZMA (500 nM) for 48 h. Data represent the mean ± s.d. of three independent experiments and were analyzed by one-way ANOVA with multiple comparisons, followed by Dunnett post hoc test for significance versus Control, **p < 0.01, ***p < 0.001, ****p < 0.0001. The control was normalized to 1. (D) After co-transfected with indicated plasmids combined with GPX4-Luc plasmid, sh-CREB plasmids and a control Renilla luciferase expression vector for 48 h, 293T cells were treated with or without GZMA(500 nM) for 48 h, the relative luciferase unit (RLU) was presented as the fold activation relative to Renilla luciferase activity. Data represent the mean ± s.d. of three independent experiments and were analyzed by two-way ANOVA, followed by Dunnett post hoc test for significance versus Control. *p < 0.05, ***p < 0.01
Fig 2: GZMA modulated PDE4/PKA/CREB cascade signaling. (A) The level of cAMP was measured by Elisa according to the instruction. Data presented as the means ± s.d. of three independent experiments and were analyzed by one-way ANOVA (left panel) and two sample t test (right panel), *p < 0.05. (B) HT-29 and Caco-2 cells were serum-starved for 24 h, followed by stimulation with GZMA (500 nM) for 1 h followed by addition of with Rp-cAMPS (10 µM) for 48 h, WB was conducted to analyze the indicated protein. The band was quantified and analyzed by one-sample t test for significance, the control was normalized as 1, data represent the mean ± s.d. ***p < 0.001, **p < 0.01, ****p < 0.0001. (C) after starvation overnight, HT-29 cells were treated with GZMA for 1 h, and the total protein was collected to detect indicated protein, Data was presented as the mean ± s.d. of three independent experiments and were analyzed by one-way ANOVA (left panel) and two sample t test (right panel), **p < 0.01, ***p < 0.001. (D) Caco-2 cells were serum starved for 24 h after 80% confluence, then stimulated as indicated for 1 h. Immunoprecipitated (IP) was employed to analyze the interaction between PKA and CREB. (E) Immunofluorescence of co-localization between PKA and CREB in Caco-2 cells treated with or without GZMA for 1 h after serum starved for 24 h. scale bar = 50 μm
Fig 3: GZMA promoted intestinal epithelial cell differentiation. (A) Immunofluorescence assay was performed to detect CDX2 expression of colonic mucosa from clinical sample (Scale bar: 100 μm), quantitation was performed by Image J, and analyzed by two-sample t test, ∗∗∗p < 0:001. (B-C) Caco-2 cells were seeded onto transwell polycarbonate membranes (0.4 μm pores). Upon confluence (21 days after seeding), the cells were treated with GZMA (500 nM) for up to 48 h. The permeability of the monolayer to FITC-dextran (4 kDa) was assessed by measuring the fluorescence intensity in the bottom chamber at Ex/Em = 485/535 nm. Data was displayed as means ± s.d. of three independent experiments and analyzed by two-sample t test for significance, ***p < 0.001.(D) Real-time PCR and (E-F) western blotting as well as (G) immunofluorescence staining were conducted to analyze the indicated gene expression at mRNA and protein level in HT-29 and Caco-2 cells treated with or without GZMA (500 nM) for 48 h (Scale bar: 25 μm), Data was showed as the means ± s.d. of three independent experiments and quantified by one-sample t test for significance, ***p < 0.001, **p < 0.01, *p < 0.05. (H) intestinal crypt isolated from mice was used to explore the effect of GZMA (500 nM) on intestinal organoid generation, microscopic examination of organoids was employed to calculate the proportion of budding organoids among every average 100 organoids. Data was exhibited as means ± s.d. of three independent experiments and analyzed by two sample t test, **p < 0.01 (Scale bar: 50 μm). (I) western blotting was conducted to analyze the indicated proteins in Caco-2 cells after transferred with sh-CDX2 plasmid, followed by stimulation with GZMA (500 nM) for 48 h, with β-actin serving as the internal control. (J-K) CD8+CD39+ T cell subsets isolated from peripheral blood of healthy donors were cultured in medium for 24 h was collected to co-culture with Caco-2 cells for 48 h combined with or without GZMA antibody supplementation. The total lysate was harvested to detect indicated proteins, Data was displayed as mean ± s.d. of three independent experiments and analyzed by one-sample t test for significance, ***p < 0.001, **p < 0.01
Fig 4: Decreased GZMA in patients with IBD and DSS-induced colitis. (A-B) The level of GZMA in serum and colonic mucosa were measured by ELISA according to the instruction. Data was displayed as the means ± s.d. of three independent experiments and analyzed by two-sample t test for significance, ***p < 0.001, **p < 0.01. (C) Real-time PCR was employed to assess colonic GZMA and IL-6 mRNA level in indicated group. Data was exhibited as the means ± s.d. of three independent experiments and analyzed by one-sample t-test for significance, ****p < 0.0001. (D) Immunofluorescence assay was performed to detect CD8, CD39, and GZMA expression in indicated group (Scale bar: 100 μm)
Fig 5: GZMA improved DSS-induced colitis in vivo (A) Representative colon length and (B) body weight changes in indicated group were measured and analyzed by one way ANOVA, The body weight changes were expressed as the percentage of initial body weight at the start of the experiments as 100%, *** p < 0.001, ** p < 0.01. (C) Immunofluorescence was performed to detect GPX4, xCT and phosphorylation of PDE4 expression in indicated group. (D) Schematic model of GZMA regulating IEC differentiation through ferroptosis
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