pe conjugate anti dpp4 antibody  (Sino Biological)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: CBX4 Regulates Replicative Senescence of WI-38 Fibroblasts

doi: 10.1155/2022/5503575

Figure Lengend Snippet: The timecourse of senescence marker expression with the serial culture of the WI-38 replicative senescence cell model system. Human diploid fibroblast WI-38 cells were profiled for senescence markers by flow cytometry and qPCR. Each serial culture is represented in a different color; the trend-line for expression of each marker was created by a smoothing spline function with 5 knots. (a) Serial culture of WI-38 was performed from CPD 37 to senescent status (CPD 56–60) for a total of 5 times. Doubling time increased during the serial culture. (b) and (c) SA- β -gal activity, protein expression of p21, p16, DPP4, and EdU proliferation markers detected by flow cytometry in proliferating (CPD 40-42, dark green) and senescent (CPD 56-60, blue) WI-38, both individually as single markers (b) or combined (c). Compared to proliferating WI-38, senescent WI-38 expressed higher SA- β -gal activity, p21, p16, DPP4 protein, and lower EdU (b). In the senescent WI-38, p16 protein was positively co-expressed with SA- β -gal activity and DPP4; SA- β -gal activity was negatively correlated with the proliferation marker EdU (c). (d) and (f) Serial culture of WI-38 from proliferating to senescent status was done repeatedly to characterize the timecourse of senescence marker development by flow cytometry and qPCR. There are no qPCR equivalents for SA- β -gal activity or EdU; however, Lamin B1 (LMNB1) was quantified by qPCR to provide a gene expression representation of cell proliferation and senescence. (e) and (g) The associations of senescence markers and genes with CPD were evaluated by repeated measures correlation (Rmcorr). The Rmcorr correlation efficient ( r rm ) of CPD with senescence markers (% of total cells) and genes (expression ratio) is depicted for analyses adjusted for repeated measurements. Green: negative correlation. Red: positive correlation.

Article Snippet: PE-conjugated anti-DPP4 mAb (2 : 100, Thermo Fisher, 12-0269-42) was added for 30 mins at room temperature.

Techniques: Marker, Expressing, Flow Cytometry, Activity Assay, Gene Expression

Journal: Oxidative Medicine and Cellular Longevity

Article Title: CBX4 Regulates Replicative Senescence of WI-38 Fibroblasts

doi: 10.1155/2022/5503575

Figure Lengend Snippet: Activation of CBX4 reduced cellular senescence markers in WI-38. CBX4 was activated using the CRISPR/Cas9 Synergistic Activation Mediator (SAM) lentiviral particle system in presenescent WI-38 (CPD47-50). (a) Representative Western blot of CBX4 protein expression in CBX4 activation (ACT) and lentiviral control (Ctrl). (c) CBX4 protein ( n = 6) and gene expression ( n = 5) both increased in response to CBX4 ACT compared with Ctrl (Ctrl: white, CBX4 ACT: light red). (c) SA- β gal, p16, and EdU proliferation expression in Ctrl and CBX4 ACT in representative flow cytometry. (d) By flow cytometry, CBX4 activation resulted in decreased protein expression of senescence markers SA- β gal, p16, and DPP4 and nonsignificantly increased EdU ( n = 7).

Article Snippet: PE-conjugated anti-DPP4 mAb (2 : 100, Thermo Fisher, 12-0269-42) was added for 30 mins at room temperature.

Techniques: Activation Assay, CRISPR, Western Blot, Expressing, Control, Gene Expression, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: CBX4 Regulates Replicative Senescence of WI-38 Fibroblasts

doi: 10.1155/2022/5503575

Figure Lengend Snippet: Regulation of senescence-related genes by CBX4. A selected panel of senescence-related genes was analyzed by qPCR microarray. Two serial culture experiments were performed, and the resulting Log2FC values were computed (normalized with YWHAZ and calculated relative to the earliest CPD of serial culture). (a) and (b) Heatmaps of standardized gene expression at 10 time points of WI-38 serial culture performed in two biological replicates. (c) the Rmcorr correlation (and 95% confidence intervals) of CPD with expression (Log2 FC) of 37 senescence-related genes from serial culture. Green: positive correlation of gene expression with CPD. Red: negative correlation gene expression with CPD. Black: insignificant correlation of gene expression with CPD. (d) Heatmap of pairwise Spearman correlations of genes measured in the serial culture experiment. Based on expression pattern and consistent with (a) and (b), two groups of genes were highly clustered. (e) Volcano plot of gene expression changes in response to CBX4 KD vs. Ctrl showing increased expression of DPP4, CDKN2A, and SLC52A1 and decreased expression of CXCL8, PCNA, DNMT1, E2F1, and PARP1. (f) Volcano plot of gene expression changes in response to CBX4 ACT vs. Ctrl showing decreased expression of DPP4, CDKN2A, and HDAC1 and increased expression of CBX4, SIRT1, and MDM4. Red: increased expression, green: decreased expression.

Article Snippet: PE-conjugated anti-DPP4 mAb (2 : 100, Thermo Fisher, 12-0269-42) was added for 30 mins at room temperature.

Techniques: Microarray, Gene Expression, Expressing