Journal: Aging Cell
Article Title: A Novel Human Cellular System for Studying Normal Aging and for Anti‐Aging Discovery
doi: 10.1111/acel.70352
Figure Lengend Snippet: The hTSC‐STB system enables simple, fast, and quantitative evaluation of known anti‐aging molecules. (A) Western blot analysis of hCG levels in hTSCs and STBs on D2, D4, and D6 treated with different concentrations of Rapamycin (RAPA), INK128, and Fisetin. These known anti‐aging molecules are able to prevent, to some extent, hTSCs from developing into fully mature STBs; (B) Western blotting analysis showed significantly decreased γH2AX in Rapamycin, INK128, and Fisetin treated STB cells, whereas the treated STBs express higher HP1γ, Lamin B1 and Ki67 than non‐treated STBs; (C) Schematic graph of the CGA‐T2A‐H2B‐EGFP reporter allele where the T2A‐H2B‐EGFP cassette is inserted into the CGA locus for fusion peptide production and subsequent cleavage into CGA and H2B‐EGFP; (D) The CGA‐EGFP hTSC reporter cells (left) efficiently differentiate into STBs (right) with particularly high EGFP signals in the nucleus (live cells); (E) Sum intensity of EGFP gradually increases in CGA‐EGFP hTSC reporter cells to STBs. EGFP signals appear to have reached a plateau on D4 as the signals are similar in D4 and D6 cells; (F) Representative immunostaining images of CGA‐EGFP hTSCs and CGA‐EGFP STB cells of D2, D4, D6 showing EGFP signals co‐stained with DAPI; (G, H) Using the CGA‐EGFP reporter hTSCs to quantitively evaluate known anti‐aging molecules. The treatments start on day 0 of hTSCs to STBs development; Normalized sum GFP intensity refers to DAPI signals. The graphs in (G) are mean ± SEM; (I) An example of a screened 96‐well library plate. Rapamycin and INK128 were used as the positive controls. Magenta rectangle marked the potential candidates (Anti‐aging and Pro‐aging); (J) Quantification of EGFP signals of the 96‐well plate in Figure . The sum intensity was normalized referring to DAPI signals; (K) β‐gal staining of STBs (D6) in the presence of candidate molecules (Anisomycin and Nitidine) in its development from hTSCs to STBs; (L) Quantification of β‐gal staining cell in (K); (M) Representative β‐gal staining images of HFF‐1 cells treated with DMSO, VP‐16, VP‐16 + INK128 25 nM, VP‐16 + Anisomycin 25 nM, and VP‐16 + Nitidine 25 nM; (N) Quantification of β‐gal staining cell in (M). One‐way ANOVA with Dunnett's test was used in statistical analysis. Ns, p > 0.05; * p ≤ 0.05, ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. All experiments have been independently repeated three times. Scale bars, 10 μm.
Article Snippet: Etoposide (VP‐16) , MedChemExpress , HY‐13629.
Techniques: Western Blot, Immunostaining, Staining
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