Figure 3
Figure 3. SENEX activates the p16 senescence pathway. (A) HUVECs were infected with EV (□) or SENEX (■) containing adenovirus. Photographs were taken at 24-hour time intervals for 4 days. The number of senescent cells, based on an enlarged morphology, were counted and presented as a percentage of total cells counted. At least 1000 cells were counted for each of the 3 individual HUVEC lines. The mean ± SEM is shown. **P < .01 and ***P < .001 compared with EV. (B) EV (□) and SENEX (■) overexpressing cells were harvested after 24, 48, and 72 hours of culture and stained for SA-β-gal expression. At least 1000 cells on each day were counted. The percentage of cells positive ± SEM is given for 3 different HUVEC lines analyzed. *P < .05 compared with EV. (C) EV and SENEX (S) overexpressing cells were harvested after 24, 48, and 72 hours of culture, and telomere length was measured by Southern blot analysis. (D) Replicative senescence in HUVECs was induced through constant passaging and the cells were harvested when the senescent morphology was evident in the majority of cells. mRNA expression was measured by Q-RT-PCR of COX2, IL-1α, PAI-1, and SENEX and standardized to cyclophilin A. The mean ± SEM of 6 replicates from 2 lines of HUVECs is shown. Early-passage cells (□) and late-passage senescent cells (■). *P < .05, **P < .01, and ***P < .001 compared with early passage. (E) SENEX (■) and EV (□) infected cells were harvested when at least 50% senescence was seen and mRNA levels determined for PAI-1, COX-2, and IL-1α by Q-RT-PCR. There were no significant differences seen between the EV and SENEX groups. This is a representative experiment of 2. (F) HUVECs were infected with SENEX or EV adenovirus for 24 and 72 hours. Total RNA was extracted and Q-RT-PCR used to determine levels of p16 standardized to Cyclophilin A. This is a representative of 4 experiments. Results are the mean ± SEM of 3 replicates of each group. *P < .05 compared with EV. (G) Total protein from 24-hour overexpressing SENEX or EV control cells was used for Western blotting using p16 or phosphorylated Rb specific antibodies. β-actin was used as a loading control. This is a representative of 3-4 experiments performed using different EC isolates.

SENEX activates the p16 senescence pathway. (A) HUVECs were infected with EV (□) or SENEX (■) containing adenovirus. Photographs were taken at 24-hour time intervals for 4 days. The number of senescent cells, based on an enlarged morphology, were counted and presented as a percentage of total cells counted. At least 1000 cells were counted for each of the 3 individual HUVEC lines. The mean ± SEM is shown. **P < .01 and ***P < .001 compared with EV. (B) EV (□) and SENEX (■) overexpressing cells were harvested after 24, 48, and 72 hours of culture and stained for SA-β-gal expression. At least 1000 cells on each day were counted. The percentage of cells positive ± SEM is given for 3 different HUVEC lines analyzed. *P < .05 compared with EV. (C) EV and SENEX (S) overexpressing cells were harvested after 24, 48, and 72 hours of culture, and telomere length was measured by Southern blot analysis. (D) Replicative senescence in HUVECs was induced through constant passaging and the cells were harvested when the senescent morphology was evident in the majority of cells. mRNA expression was measured by Q-RT-PCR of COX2, IL-1α, PAI-1, and SENEX and standardized to cyclophilin A. The mean ± SEM of 6 replicates from 2 lines of HUVECs is shown. Early-passage cells (□) and late-passage senescent cells (■). *P < .05, **P < .01, and ***P < .001 compared with early passage. (E) SENEX (■) and EV (□) infected cells were harvested when at least 50% senescence was seen and mRNA levels determined for PAI-1, COX-2, and IL-1α by Q-RT-PCR. There were no significant differences seen between the EV and SENEX groups. This is a representative experiment of 2. (F) HUVECs were infected with SENEX or EV adenovirus for 24 and 72 hours. Total RNA was extracted and Q-RT-PCR used to determine levels of p16 standardized to Cyclophilin A. This is a representative of 4 experiments. Results are the mean ± SEM of 3 replicates of each group. *P < .05 compared with EV. (G) Total protein from 24-hour overexpressing SENEX or EV control cells was used for Western blotting using p16 or phosphorylated Rb specific antibodies. β-actin was used as a loading control. This is a representative of 3-4 experiments performed using different EC isolates.

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