..................................................
Figure 1 A 24 hour pre-treatment with thymidine enhances gene repair levels. Various concentrations of thymidine were added to a growing culture of DLD-1 cells 24 hours prior to the introduction of 1 μM oligonucleotide (EGFP3S/47NT). Cells were allowed to recover for 48 hours after electroporation and were harvested for FACS analysis. Correction efficiency is reported as the percent of live green fluorescent cells in the total cell population (50,000 per experiment); mean and standard deviations were calculated based on values obtained from 3–4 samples per treatment. T-tests were performed comparing the 1 mM and 8 mM thymidine pre-treatment to the 0 mM sample (***p -5).
..................................................
Figure 2 Pre-treatment with thymidine arrests cells in G1 and once removed, allows a synchronous entry into S phase. (A) Thymidine was added at 1 mM (ii) or 0 mM (i) for 24 hours, cells were then harvested and processed for cell cycle analysis, representing the cell cycle profile at the time of oligonucleotide electroporation. (B) Cells were treated for 24 hours as in A, but were washed 2× with PBS at 24 hours and released into fresh media for 8 hours, at which time they were either collected for cell cycle analysis (i and ii) or were stained for BrdU incorporation (iii and iv) to measure their replicative state. The position of M1 (BrdU+ marker) was determined by a BrdU(-) control, not shown, and percents indicate the number of cells that are positive for BrdU in the total sample size (25,000 cells).
..................................................
Figure 3 Over time, gene repair levels diminish regardless of thymidine pre-treatment. Cells were treated either with no thymidine (a and b) or with 1 mM thymidine (c and d) for 24 hours prior to electroporation of the oligonucleotide. The cells were then allowed to recover for 48 hours (a and c) or 144 hours (b and d), at which points they were harvested and measured by FACS for eGFP expression. The correction efficiency reports the percent of the total number of cells (50,000 in each sample) that are live and positive for eGFP fluorescence. The mean and standard deviations were calculated based on the results from 4 individual samples of each treatment/time point; T-tests were performed comparing samples a to b and c to d (***p -4).
..................................................
Figure 4 Combining the 1 mM thymidine pre-treatment with a 2 mM post-treatment retains high levels of gene repair. Cells were treated with 1 mM thymidine for 24 hours, electroporated with 1 μM EGFP3S/47NT oligo, and recovered in complete medium without thymidine (a and b) or in the presence of 2 mM thymidine (c through f) for 48 hours (a and c) or for 144 hours (b and d-f). At 48 and 72 hours, sample e and f, respectively, were washed 2× with PBS and fresh medium was added that did not contain any thymidine (termed "w/o" for "washed out"); these cells were collected at 144 hours and analyzed by FACS for eGFP fluorescence. Correction efficiency depicts the percentage of live green fluorescent cells in the total number of cells counted (50,000 per sample); means and standard deviations were calculated based on the results of three to four independent experiments. T-tests were performed comparing b to d and e (**p
..................................................
Figure 5 Prolonged exposure to thymidine results in cellular senescence. (A) Following a 1 mM thymidine 24 hour pre-treatment, cells were electroporated and released into complete medium (1-0 mM) or medium supplemented with 2 mM thymidine (1–2 mM). At 48, 72 and 144 hours, cells were stained for the senescent marker β-galactosidase. The four quadrants for the 1–2 mM sample at 48 hours are combined images obtained from four different fields of view, due to the low confluency of the sample. (B) Images depict varying views and different wells of the 1–2 mM thymidine treated cells at 144 hours. All images are at 20× magnification and counts of individual cells in a representative field were performed to evaluate the degree of senescence induction.
..................................................
Figure 6 Removal of the 2 mM thymidine post-treatment at 48 hours, but not at 72 hours, prevents cellular senescence. Cells were treated with 1 mM thymidine prior to electroporation and then recovered in 1 mM thymidine. At 48 hours and 72 hours, as indicated, cultures were washed 2× with PBS and fresh medium was added ("w/o": washed out). At 144 hours, cells were assayed for senescence-associated β-galactosidase activity. All images are at 20× magnification.
..................................................
Figure 7 Cells are able to resume replication after the removal of 2 mM thymidine during recovery. Cells were treated with 1 mM thymidine for 24 hours, electroporated, and recovered in 2 mM thymidine (1–2 mM) or without thymidine (1-0 mM). At 48, 72, and 144 hours, the culture media was supplemented with BrdU and cells were later assayed by FACS for its incorporation. The 1-0 mM culture reached confluency by 96 hours and was passaged once prior to the 144 hour time point. The w/o ("wash out") samples received 2 mM thymidine in the recovery phase, which was subsequently washed out at 48 and 72 hours, respectively, and later assayed at 144 hours for BrdU incorporation. Position of M1 (BrdU+ marker) was determined by a BrdU(-) control, not shown, and percents indicate the number of cells that are positive for BrdU incorporation out of the total population (25,000 cells).
..................................................
Figure 8 Following the removal of 2 mM thymidine from the post-treatment, cells are able to resume cell division at rates comparable to no treatment samples. Following a 24 hour 1 mM pre-treatment with thymidine, cells were collected and stained with the cell-membrane dye, PKH26, and then released into medium supplemented with 2 mM thymidine (iv and v) or complete medium (ii and iii). Fluorescence of the PKH26 dye was measured in a subset of the cells immediately following staining and prior to re-plating to obtain an initial fluorescence value (i: T0); subsequent samples were removed at 48 (ii and iv) and 144 hours (iii and v) following re-plating for the same analysis. Sample (v) was washed 2× with PBS at 48 hours, received fresh medium and analyzed at 144 hours. Values indicate the mean fluorescence intensity (m.f.i.) of the population at each time point (shaded peak); non-shaded graph represents the fluorescence levels at T0 and filled in graphs represent the fluorescence intensity at the indicated times. M1 is the boundary for the PKH26(-) samples and M2 indicates PKH26(+) fluorescence.
..................................................
Answers to all your Biology Questions
