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. 2003 Aug;23(16):5540–5555. doi: 10.1128/MCB.23.16.5540-5555.2003

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Copyright © 2003, American Society for Microbiology

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FIG. 2. — (A) ΔNp73 expression rescues primary MEFs from spontaneous cellular senescence. Morphology of MEFs of the indicated genetic background and infected with retroviruses expressing either vector GFP alone (left column) or ΔNp73α (right column) after 13 and 15 passages in culture. Control MEFs are senescent, whereas ΔNp73-expressing cells are not senescent. (Passage 8 is close to the end of their normal life span. After 12 to 15 passages, ΔNp73-expressing MEFs reproducibly produce numerous immortalized colonies that can be maintained in culture indefinitely.) (B to E) ΔNp73 expression promotes cellular immortalization. (B) Long-term growth curves of 129 MEFs infected with MSCV/IRES/GFP retroviruses expressing GFP only (•), tetramerization-defective ΔNp73 L322P (▵), ΔNp73 (○), or dominant-negative p53 R175H (▴). Cells were passaged in a 3T3 protocol. Total cell numbers per 60-mm culture dish were determined every 3 days prior to redilution for the next passage. The arrow marks the time point when numerous colonies appeared on the plates, each of which could be readily established as immortal cell lines. (C and D) Experiments similar to those in panel B with 129 MEFs (C) and 129×B6 MEFs (D) after infection with the indicated retroviruses and passage in a 3T3 protocol. Growth kinetics are plotted as the fold increase of total cell numbers after each passage. The top of the panel compares GFP vector (•) and ΔNp73 (○ and □ in panel D); the bottom of the panel compares GFP vector (•), ΔNp73 (○ and □ in panel D), p53 R175H (▵), and p53^−/− (▴ in panel C). I and II refer to different populations of immortal ΔNp73 MEFs assayed in parallel. (E) A GFP-FACS analysis to determine spontaneous self-selection for high ΔNp73 expression over time is shown in the top part of the panel. Freshly MSCV/IRES/GFP-transduced 129 MEFs (passage 4) showed a ΔNp73 infectivity of 75% as assessed by GFP, whereas the same culture 11 passages later showed a ΔNp73 infectivity close to 100%. Curves: gray, parental MEFs used as a reference; black, ΔNp73-infected MEFs. At the bottom of the panel is a Western blot analysis with anti-p73 antibody after ΔNp73 expression in pooled MEFs over time. The lanes show GFP control MEFs (lane 1) and ΔNp73-infected MEFs 3 days after initial transduction (lane 2) and after spontaneous immortalization at passage 15 (lanes 3 and 4; two independent clones are shown). Erk was used as a loading control.

FIG. 2. — (A) ΔNp73 expression rescues primary MEFs from spontaneous cellular senescence. Morphology of MEFs of the indicated genetic background and infected with retroviruses expressing either vector GFP alone (left column) or ΔNp73α (right column) after 13 and 15 passages in culture. Control MEFs are senescent, whereas ΔNp73-expressing cells are not senescent. (Passage 8 is close to the end of their normal life span. After 12 to 15 passages, ΔNp73-expressing MEFs reproducibly produce numerous immortalized colonies that can be maintained in culture indefinitely.) (B to E) ΔNp73 expression promotes cellular immortalization. (B) Long-term growth curves of 129 MEFs infected with MSCV/IRES/GFP retroviruses expressing GFP only (•), tetramerization-defective ΔNp73 L322P (▵), ΔNp73 (○), or dominant-negative p53 R175H (▴). Cells were passaged in a 3T3 protocol. Total cell numbers per 60-mm culture dish were determined every 3 days prior to redilution for the next passage. The arrow marks the time point when numerous colonies appeared on the plates, each of which could be readily established as immortal cell lines. (C and D) Experiments similar to those in panel B with 129 MEFs (C) and 129×B6 MEFs (D) after infection with the indicated retroviruses and passage in a 3T3 protocol. Growth kinetics are plotted as the fold increase of total cell numbers after each passage. The top of the panel compares GFP vector (•) and ΔNp73 (○ and □ in panel D); the bottom of the panel compares GFP vector (•), ΔNp73 (○ and □ in panel D), p53 R175H (▵), and p53^−/− (▴ in panel C). I and II refer to different populations of immortal ΔNp73 MEFs assayed in parallel. (E) A GFP-FACS analysis to determine spontaneous self-selection for high ΔNp73 expression over time is shown in the top part of the panel. Freshly MSCV/IRES/GFP-transduced 129 MEFs (passage 4) showed a ΔNp73 infectivity of 75% as assessed by GFP, whereas the same culture 11 passages later showed a ΔNp73 infectivity close to 100%. Curves: gray, parental MEFs used as a reference; black, ΔNp73-infected MEFs. At the bottom of the panel is a Western blot analysis with anti-p73 antibody after ΔNp73 expression in pooled MEFs over time. The lanes show GFP control MEFs (lane 1) and ΔNp73-infected MEFs 3 days after initial transduction (lane 2) and after spontaneous immortalization at passage 15 (lanes 3 and 4; two independent clones are shown). Erk was used as a loading control.