Fig 1: Immune-epithelial interaction in aged colon tissues.a The schematic image depicts the effect of senescent immune cells on the surrounding microenvironment of tissues. b A dot plot illustrates the incoming and outgoing strength (interaction count) in each cell type in GSE178341. c The ingoing and outgoing interaction strength across 18 different signaling pathways in each cell type. A blue box indicates the interaction strength of p16INK4A- T cells, while a red box indicates the interaction strength of p16INK4A+ T cells. The y axis of the top bar graph indicates the average number of interactions or connections for each cell type within the signaling network. d The interaction strength of PARs signaling network is displayed. e The strength of sender, receiver, mediator, and influencer in the PARs signaling pathway network were examined in each cell type. f The IHC analysis of PAR1 (left panel) and PAR2 (right panel) in normal colon tissues from young and elderly individuals is shown. g The violin plot displays the mRNA expression level of GzmA in p16INK4A- and p16INK4A+ T cells from GSE178341 (left panel). The violin plot illustrates the mRNA expression of GzmA in T cells from young and old individuals (right panel). h The IHC analysis of GzmA was performed in colon tissues from young and old individuals, respectively (left panel). The right panel shows the quantification data. The data is presented as mean ± standard deviation. “Young” and “Old” indicate the young and the elderly individuals, respectively. The p-value is calculated using Mann–Whitney U test. i The multiplex IHC analysis shows the expression of CD3 (brown) and GzmA (red) (upper panel) and p16INK4A (brown) and GzmA (red) (lower panel) in old individuals, respectively. j The multiplex IHC analysis shows the expression of GzmA (brown) and PAR1 (red) (upper panel) and GzmA (brown) and PAR2 (green) (lower panel) in old individuals, respectively.
Fig 2: The interaction between senescent T cells and epithelial cells in vitro.a A schematic image illustrating how GzmA affects colonic epithelial cells through PARs signaling. b Overall scheme of the T cell isolation and senescence process (upper panel). Isolated pan-T cells after stimulation with anti-CD3/28 cocktail and rhIL-2 were assessed for cumulative population doubling level (PDL) (lower panel). Young cells were defined as 2 < cPDL < 5 and senescent cells as cPDL > 9. c The C12FDG staining analysis using flow cytometry (upper panels). The mRNA expression level of representative T cell senescence markers (p21Waf1, p16INK4A, CD28, and CD57) are shown (lower panel). ND not detected. d The mRNA (upper panel) and protein (lower panel) levels of GzmA in young and senescent T cells are performed by real-time PCR and ELISA, respectively. e The IHC analysis of cleaved caspase-3 in normal colon tissues from young and elderly individuals (upper panel) and 4-month and 18-month-old mouse colon tissues (lower panel). The right panels display the quantification data, respectively. f,g Human primary colon epithelial cells were co-cultured with young or senescence T cells (f) or treated with CM of young or senescence T cells (g) for 3 days. The luminescence-based assay and ICC to determine cleaved caspase-3/7 activity and cleaved caspase-3 expression level were performed, respectively. h IHC analysis for IL8 was performed in normal colon from young and elderly tissues (upper panel). The IHC analysis results are categorized into “Low,” “Moderate,” and “High” based on the intensity of the immunostaining, with representative images provided for each category. The left lower panel shows the quantification data for IHC analysis. The right lower panel shows a dot plot illustrating the expression of CXCL8 (IL8) in colonic epithelial cells from young and elderly individuals in the GSE178341 data set. i The CXCL8 (IL8) mRNA expression was analyzed by real-time PCR in human colon epithelial cells, which were co-cultured with young or senescent T cells (upper panel) and treated with CM of young or senescent T cells (lower panel) for 3 days. A p-value in (h) is calculated using the Chi-square test. The rest of the p-values are calculated using the Mann–Whitney U test. The graph in (b) is shown as mean ± standard deviation, while the rest of the bar graphs are shown as mean + standard deviation. “Young” and “Sen” in (c,d,f,g,i) indicate the young and senescent T cells, respectively. “Young” and “Old” in (e,h) indicate the young and the elderly individuals, respectively.
Supplier Page from Abcam for Recombinant Human Granzyme A protein