Research Paper Volume 4, Issue 10 pp 664—673

Figure 1. CBS depletion reduces the rate of cell proliferation. (A) Protein was isolated from HUVEC and HDF in passage 6 (young) and subsequent passages as indicated; HUVEC reached senescence at passage 25, whereas HDF reached senescence at passage 27. CBS levels were determined by Western blot analysis, GAPDH served as loading control. (B) Left panels: CBS gene expression was inactivated by lentiviral shRNA vectors in early-passage HUVEC and HDF, as indicated. Cumulative population doublings (cPDL) were calculated, beginning 12 days after infection. Growth curves represent three independent experiments in each case. Right panels: CBS knockdown was verified by Western blot. (C) Bar diagram displaying relative percentage of BrdU positive (= proliferating) HUVEC 42 days after infection with lentiviral shRNA vectors. The percentage of BrdU positive cells was determined using flow cytometry; data are represented as mean ± SE (n=3). ***: p<0.001. (D) Bar diagram displaying relative percentage of annexin V positive (= apoptotic) HUVEC 42 days after infection with lentiviral shRNA vectors. The percentage of apoptotic cells was determined using flow cytometry; data are represented as mean ± SE (n=5). n.s: not significant