Fig. 4

TERT improved GDF11-mediated rejuvenation of old CD133⁺/VEGFR2⁺ cells. a The number of cumulative population doublings of old CD133⁺/VEGFR2⁺ cells in the presence or absence of rhGDF11 or siGDF11 under long-term hypoxic culture. b–d The number of cumulative population doublings (b), FACS analysis of annexin V/propidium iodide (c), and quantification of the senescence assays using β-galactosidase staining (d), were determined in old CD133⁺/VEGFR2⁺ cells receiving rhGDF11 or siGDF11 under long-term hypoxic culture. All data represent means ± SD. p < 0.05: *vs. vehicle, ^†vs. rhGDF11 adding, and ^‡vs. siGDF11 adding (n = 10 per group). Afterward, old CD133⁺/VEGFR2⁺ cells were transfected with vectors encoding TERT (adTERT) or TERT siRNA (siTERT) in the presence or absence of recombinant human GDF11 (rhGDF11) under hypoxic conditions. e, f qRT-PCR analysis of the mRNA and protein expression of GDF11 and TERT, respectively, under various conditions. g Western blot analysis of protein expression of GDF11 and TERT under various conditions. h Differences in telomerase activity. i GDF11 and TERT expression in cells determined by immunofluorescence with anti-GDF11 (red) and anti-TERT (green) antibodies, respectively. Also shown are DAPI staining (nuclei; blue) and merged images. Scale bars = 50 µm. j Cell death was evaluated via FACS analysis of annexin V/propidium iodide. k Cell proliferation was assessed by FACS analysis of Ki-67-positive cells, showing the greatest positive staining in the rhGDF11 + adTERT group, the second highest in the group receiving adTERT alone, and the leatest in the group treated with siTERT. l Number of cumulative population doublings under long-term hypoxic culture. m Quantification of the senescent assays using β-galactosidase staining. All data represent means ± s.e.m. p < 0.05: *vs. vehicle, ^†vs. rhGDF11 adding, ^‡vs. adTERT adding, ^§vs. siTERT adding, and ^||vs. rhGDF11 and adTERT adding (n = 10 per group)