Journal: Experimental & Molecular Medicine
Article Title: Distribution and impact of p16 INK4A+ senescent cells in elderly tissues: a focus on senescent immune cell and epithelial dysfunction
doi: 10.1038/s12276-024-01354-4
Figure Lengend Snippet: 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 C 12 FDG staining analysis using flow cytometry (upper panels). The mRNA expression level of representative T cell senescence markers (p21 Waf1 , p16 INK4A , 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.
Article Snippet: These mixtures were treated to human colonic epithelial cells (HCoEpiC, Cell Biologics, Inc.) for 1 day.
Techniques: Cell Isolation, Isolation, Staining, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Luminescence Assay, Activity Assay, Immunostaining, MANN-WHITNEY, Standard Deviation
