Fig 1: Altered Dynamics of the SENP7 Interactome in DSS Mice(A) Coomassie staining (left panel) and immunoblotting (right panel) of samples co-immunoprecipitated with anti-SENP7 or IgG from colonic lysates of control and DSS7 mice. Samples from (A) were in-gel digested and analyzed by ESI-tandem mass spectrometry (MS/MS) (n = 5 mice).(B) Venn diagram displaying the number of individual proteins identified by MS/MS.(C) The relative abundance of various signaling pathways based on MS/MS prepared using the Reactome database (shown as that of DSS-7 mice versus control). Gene Ontology is represented as fold enrichment. Probability was determined using a binomial statistic for the false discovery rate (FDR) and a p value cutoff of ≤ 0.05 significance level.(D) Protein-protein interaction network for SENP7 and its interacting hub proteins based on the Boolean network model. The size of the node (circles) in the network represents the fold change identified by the LFQ algorithm, and the border of the nodes represents iBAQ-based fold change.(E) Schematic of the steps involved in co-culture experiments using CT26 cells and total immune cells from MLNs.(F) Population frequency γδ T cells was calculated and plotted (labeling: immune cells, cells from MLNs; immune cells + IL15, IL-15 treatment of cultured immune cells for 3 days; C1, immune cells co-cultured with CT26 cells transfected with empty FLAG vector; SENP7 WT O/E, SENP7 wild-type overexpressed CT26 in co-culture setup; SENP7 C992A O/E, catalytic mutant of SENP7; SENP7 WT O/E + anti-IL15, SENP7 overexpressed epithelial cells with IL-15 neutralization).(G) ELISA for indicated cytokines from the supernatant of C1, SENP7 WT O/E, SENP7 WT O/E + Anti-IL15, and SENP C992A O/E cells described above. Each plot is representative of three independent experiments. Results are represented as mean+ SEM.p values were determined by Student’s t test and one-way ANOVA with Tukey’s posttest (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant). See also Figure S2 and Tables S1, S2, and S3.
Fig 2: Epithelial SENP7 Overexpression Triggers γδ T Cell Activation and Inflammation(A) Schematic representation of co-culture experiments involving CT26 cells and total immune cells.(B–F) Cytokines analysis from culture supernatant by ELISA for (B) IFN-γ, (C) TNF-α, (D) IL-10, (E) IL-17, and (F) TGF-β (C3, monoculture epithelial cells transfected with empty FLAG vector; C4, scrambled siRNA transfected monoculture; C5, empty-FLAG-vector-transfected epithelial cells with a γδ T cell; C6, scrambled siRNA transfected co-cultured epithelial cells with a γδ T cell; C7 and C8, epithelial cells with empty FLAG vector and scrambled siRNA co-cultured with total immune cells from MLNs. Each bar is representative of three independent experiments with three technical replicates. Data are presented as mean + SEM.(G) Percentage of IFN-γ - and IL-17A-secreting γδ-TCR+ cells. Cultured immune cells from MLN or spleen of indicated group were surface stained for anti-γδTCR antibody followed by intracellular staining for anti-IL-17A and anti-IFN-γ (n = 3 mice).(H) Fluorescence immunostaining of SIAH2 (green) and γδ T cells (red) in colonic sections of untreated and DSS7-treated mice. Scale bar, 100 um. Right panel represents mean intensity values of SIAH2 and number of γδ T cells.(I) ELISA of IL-15 and KGF from mucosal extracts of UC (n = 8), CD (n = 8), and control (n = 5).(J and K) Correlation analysis of SIAH2 and SENP7 expression with disease severity. Mild and moderate groups are divided on the basis of disease activity index (20 mild cases and 18 moderate cases). For flow cytometry, cells were acquired on a BD FACS Canto, analyzed using FlowJo, and represented in the form of a bar graph (mean + SEM values). Statistical differences were calculated using Prism by two-way ANOVA with Tukey’s test.*p < 0.05; **p < 0.01; ****p < 0.001. See also Figure S7.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse IL-15 Protein