Fig. 1: Design, synthesis and validation of WOS-Cy7Gal for SA-β-Gal activity detection. | Nature Communications

Fig. 1: Design, synthesis and validation of WOS-Cy7Gal for SA-β-Gal activity detection.

From: A renal clearable fluorogenic probe for in vivo β-galactosidase activity detection during aging and senolysis

Fig. 1: Design, synthesis and validation of WOS-Cy7Gal for SA-β-Gal activity detection.The alternative text for this image may have been generated using AI.

a Synthetic route used for the preparation of the WOS-Cy7Gal probe. b X-Gal histochemical staining (left) and confocal images of WOS-Cy7Gal (right) in hUVECs, treated with the senescence-inducing drug palbociclib (senescent) or not (control), for the determination of SA-β-Gal activity. c Phenotypic characterization of senescence in hUVECs by immunocytochemical detection of senescence-associated markers (n = 3 control and senescent cells). Note that senescent hUVECs showed a marked decrease in proliferation with lower Ki67 levels, a significant increase in the levels of p21 and DNA damage foci γH2AX, as well as a loss of lamin B1. d Quantification of WOS-Cy7Gal-associated median fluorescence intensity (MFI) in control and senescent hUVECs by flow cytometry (n = 3 control and senescent cells). The fold change refers to control hUVECs. e Confocal images of control and senescent hUVECs labeled with lamin B1 and WOS-Cy7Gal. Notice that senescent cells displayed lower fluorescence levels of lamin B1 and higher of the Cy7 fluorophore, compared to control ones. f Confocal images of control and senescent hUVECs labeled with p16 and WOS-Cy7Gal. Observe that senescent cells showed a brighter signal for both markers compared to control ones. g Flow cytometry histogram of the MFI of probe-released Cy7 and of p16, comparing control and senescent hUVECs. h Percentage of p16-/low and p16+/high senescent cells and flow cytometry histogram of WOS-Cy7Gal-associated MFI within these two cell populations. It is worth noting that the highest levels of fluorescence related to WOS-Cy7Gal corresponds to p16+/high cells. The graphs show the mean ± SEM. Unpaired and paired Student’s two-tailed t-tests were used for statistical analysis in graphs c and d, respectively. The number of independent biological samples (represented as dots) used and the exact p-values are indicated in the graphs. Scale bars: 100 μm. Source data is provided as a Source Data file.

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