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. Author manuscript; available in PMC: 2017 Aug 7.
Published in final edited form as: Nat Med. 2016 Mar 7;22(4):412–420. doi: 10.1038/nm.4054

Figure 6.

Figure 6

p16-induced senescence of human cells leads to enhanced GSIS. (a) FACS analysis of Ki67-stained EndoC-βH2 cells 3 weeks after infection with a lentivirus expressing GFP (blue line) or Cre + GFP(red line); GFP+ cells are shown. The experiment was done three times, (b) Representative images (out of ten taken) of EndoC-βH2 cells expressing GFP (left) or Cre + GFP (right) stained for SA–β-Gal activity (blue) in 3 weeks after infection, (c) Representative images (out of ten taken) of EndoC-βH2 cells expressing either GFP (left) or Cre + GFP (right) that were stained for insulin (red), (d) Insulin secretion levels by EndoC-βH2 cells expressing either GFP (white) or Cre+ GFP(gray) after incubation in medium containing low (2.8 mM) or high (16.7 mM) glucose concentrations for 1 h. Values are the mean of four replicates per group ± s.d., normalized to cell number and shown relative to insulin levels in control cells incubated in medium with high glucose (defined as 1). (e) FACS analyses of FSC-A, TMRE staining and 2-NBDG fluorescence in control and Cre-expressing cells. The ratios indicate the mean fluorescence value from Cre-expressing to that from control cells, (f) Representative images (out of ten taken) of SA–β-Gal activity (blue) in EndoC-βH2 cells infected with an empty vector alone (left), an empty vector followed by the Cre-expressing lentivirus (middle) or an shp16-expressing construct followed by the Cre-expressing lentivirus (right), (g) Insulin secretion levels by EndoC-βH2 cells infected with an empty vector alone, an empty vector followed by the Cre-expressing lentivirus or an shp16-expressing construct followed by the Cre-expressing lentivirus (black), analyzed as in d. Values are the mean of five replicates ± s.d. (h) FACS analysis of TMRE staining in the cells indicated in g. (i) Left, insulin secretion levels by control (GFPonly) and Cre-expressing cells treated with vehicle or the mTOR inhibitor Torin1 for 3 weeks. Right, similar analysis of control (vector only) and Cre-expressing cells treated with either vehicle or the PPAR-γ inhibitor GW9662 for 3 weeks. Values are the mean of five replicates ± s.d. (j) Schematic diagram summarizing the effects of p16-induced senescence on beta cell function. Components of the senescence program that contribute to increased insulin secretion are highlighted in red. Question mark represents potential additional effectors. Throughout, **P< 0.005, ***P< 0.0005; by Student’s t-test. Scale bars, 20 μm.