Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
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Edited by Robert F. Furchgott, State University of New York Downstate Medical Center, Brooklyn, NY, and approved May 8, 2001 (received for review March 13, 2001)
Article Figures & SI
Figures
- Figure 1
(A) T84, Caco2, or SW480 cells, stimulated with 10% FBS, were incubated for 48 h with 1 μM ST (■) or PBS (□), and then protein and/or cells were quantified as described in Materials and Methods. Values are expressed as the percentage increase in cell number or protein stimulated by FBS relative to those values at t0 (baseline). Total cells at t0 were: T84, 4.7 × 106 ± 0.2; Caco2, 1.8 × 106 ± 0.4; and SW480, 3.3 × 106 ± 0.4. T84 protein content at t0 was 0.5 ± 0.02 mg/ml. (B) T84, Caco2, and SW480 cells were synchronized 6 h after seeding by starvation for 18 h and then stimulated with 10% FBS for 24 h in the presence of 1 μM ST (■) or PBS (□). After 21 h, [3H]thymidine was added to the media, and incubation was continued for another 3 h. [3H]Thymidine incorporation into DNA was quantified as described in Materials and Methods. Values are expressed as a percentage of the [3H]thymidine incorporation stimulated by FBS in the presence of PBS. (C) T84 cells were synchronized by starvation, stimulated to proliferate by addition of 10% FBS, and exposed to [3H]thymidine and the indicated concentrations of ST, as described in B. After 24 h of incubation, [3H]thymidine incorporation into DNA was quantified as described in Materials and Methods. *, P < 0.05; **, P < 0.01.
- Figure 2
(A) T84 cells were synchronized by starvation in EMEM depleted of l-glutamine for 24 h. Proliferation was stimulated with EMEM containing 10 mM l-glutamine; cells were incubated for 48 h with 1 μM ST or PBS, and quantified as described in Fig. 1A. Values are expressed as the percentage increase in cell number stimulated by l-glutamine relative to those values at t0. Total cells: at t0 = 0.4 × 106 ± 0.07; at 48 h, control = 1.4 × 106 ± 0.2; ST = 0.9 × 106 ± 0.06. (B) Cells were synchronized and stimulated to proliferate by glutamine (as described in A), and [3H]thymidine and 1 μM ST (■) or PBS (□) was added and incubated for the indicated times. At the conclusion of incubations, [3H]thymidine incorporation into DNA was quantified as described in Materials and Methods. Values reflect a representative experiment. In B2, data obtained in B1 are expressed as {100 − [([3H]thymidine incorporation in ST-treated incubations)/([3H]thymidine incorporation in control incubations) × 100]}. *, P < 0.05; **, P < 0.01.
- Figure 3
(A) T84 cells were synchronized 6 h after seeding by starvation and then stimulated to grow by adding 10% FBS in the presence of 1 μM ST or PBS. After 24 h, cells were trypsinized, pelleted, and then fixed and stained with PI. Fluorescence analysis of DNA content was performed as described in Materials and Methods, and the percentage of cells in each phase of the cell cycle is represented. Data are from a representative experiment. After synchronization by starvation (t0), 75.7% ± 4.5 of T84 cells were in the G0/G1 phase of the cell cycle. (B1) T84 cells were synchronized by starvation in EMEM depleted of l-glutamine for 48 h, stimulated to proliferate with EMEM containing 10 mM l-glutamine, and incubated for the indicated time points with 1 μM ST (■) or PBS (□). [3H]Thymidine was added for the last 2 h of incubation, and the 3H incorporated into DNA was measured as described in Materials and Methods. Data reflect a representative experiment. Results from three experiments performed as described in B1 are presented in B2 as mean ± SEM. *, P < 0.05.
- Figure 4
(A) T84 cells were synchronized and stimulated to proliferate by l-glutamine, as described in Fig. 2A, with simultaneous addition of 1 μM ST, 1 μM uroguanylin (URO), or PBS (CTR). Incubations were continued for 24 h, and cells were trypsinized and pelleted, divided in 1 × 106 aliquots, fixed, and permeabilized by using Cytonin reagent. Biotinylated DNA was costained with both FITC-conjugated streptavidin and PI. The positive control (TACS) was generated by using the TACS-Nuclease provided with the FlowTACS kit. Flow cytometry analysis was performed as described in Materials and Methods, and data were plotted in two-dimensional format. Data are from a representative experiment. (B) Mean ± SEM of the percentage of FITC-positive T84 cells (apoptotic/necrotic) from three experiments performed as in A. (C) T84 cells (seeded at a density of 2 × 105 into 35-mm dishes) were cultured for 7 days in DMEM/F12, plus 10% FBS. Preconfluent monolayers were washed with DMEM (4.5 g/liter glucose, containing l-glutamine) and incubated in that media for 16 h. Cells were washed again in DMEM and then incubated for 2 h in that media supplemented as described in Materials and Methods (vehicle, PBS or DMSO; uroguanylin, URO). DNA was analyzed as described in Materials and Methods. The U937 positive control DNA was provided in the fragmentation analysis kit. Data are from a representative experiment.
- Figure 5
(A) Cells were synchronized, and proliferation was stimulated by l-glutamine, as described in Fig. 2A, and, 21 h later, cells were exposed to PBS or the indicated concentrations of ST (●) or uroguanylin (○). After 15 min of incubation, [3H]thymidine was added to the media, and incubation was continued for another 3 h. [3H]Thymidine incorporation into DNA was determined as described in Materials and Methods. (B) Cells were synchronized, stimulated to proliferate, and exposed to 1 μM ST (■) or PBS (□), as described in A. After 3 h of incubation, media was aspirated, and cGMP and cAMP were quantified as described in Materials and Methods. Data from a single experiment are expressed as fold accumulation in ST-treated compared with PBS-treated cells (control; CTR). (C) Cells were synchronized, stimulated to proliferate, and pulse labeled with [3H]thymidine, as described in A, and, 15 min later, T84 cells were exposed to PBS (CTR), 1 μM TJU 1-103 (TJU), 1 μM ST, 1 μM uroguanylin (URO), 5 mM 8-Br-cGMP, 10 μM zaprinast (ZAP), or 1 μM ST plus 10 μM zaprinast. After 3 h of incubation, [3H]thymidine incorporation into DNA was quantified as described in Materials and Methods. Results are the mean ± SEM of a representative experiment performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells
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