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. 2009 Dec 30;43(5):617–627. doi: 10.1165/rcmb.2008-0335OC

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Copyright © 2010, American Thoracic Society

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Figure 1. — Tsc2-null cells produce a proliferative nonadherent (N-Adh) subpopulation at confluence. (A) After confluence, Tsc2^−/− cells (EEF-8 [red] and EEF-4a [green]) proliferated faster than wild-type cells (EEF-4 [black]). Cells were manually counted at the indicated time points after confluence. Average error bars ± 0.1. (B) Cell proliferation as determined by BrdU-Hoechst flow cytometry was analyzed for subconfluent (left graph) and postconfluent (right graph) wild-type (EEF-4 [black]) and Tsc2 mutant (EEF-8 [red]; EEF-4a [green]) cells over 72 hours. BrdU quenching of Hoechst fluorescence as compared with ethidium bromide fluorescence identified actively cycling cells. The y axis represents the proportion of cells that have passed through the first cell cycle relative to the total number of cells. Average error bars ± 0.1. (C) Morphology of postconfluent cultures of Tsc2^+/+ and Tsc2^−/− fibroblasts. Arrows indicate refractile cells that have detached from the plate. (D) N-Adh Tsc2^−/− cells (8 and 4a) are viable as determined by trypan blue exclusion assays. Each cell viability assay was performed in duplicate, and the results shown represent the mean values ± SEM of three independent experiments, presented as the percentage of viable cells. In addition, replated N-Adh Tsc2^−/− cells grow in foci-like colonies (right panels). (E) N-Adh Tsc2^−/− cells undergo multiple rounds of cell proliferation as determined by BrdU-Hoechst flow cytometry. At confluence, BrdU was added to the media for 72 hours. Nonadherent cells were collected and processed for analyses. Plots depict the number of cells versus Hoechst intensity and show the distribution of cells in G0/G1, G1′, and G1" phases representing the relative proportion of cells in the first, second, and third cell cycle postlabeling, respectively.

Figure 1. — Tsc2-null cells produce a proliferative nonadherent (N-Adh) subpopulation at confluence. (A) After confluence, Tsc2^−/− cells (EEF-8 [red] and EEF-4a [green]) proliferated faster than wild-type cells (EEF-4 [black]). Cells were manually counted at the indicated time points after confluence. Average error bars ± 0.1. (B) Cell proliferation as determined by BrdU-Hoechst flow cytometry was analyzed for subconfluent (left graph) and postconfluent (right graph) wild-type (EEF-4 [black]) and Tsc2 mutant (EEF-8 [red]; EEF-4a [green]) cells over 72 hours. BrdU quenching of Hoechst fluorescence as compared with ethidium bromide fluorescence identified actively cycling cells. The y axis represents the proportion of cells that have passed through the first cell cycle relative to the total number of cells. Average error bars ± 0.1. (C) Morphology of postconfluent cultures of Tsc2^+/+ and Tsc2^−/− fibroblasts. Arrows indicate refractile cells that have detached from the plate. (D) N-Adh Tsc2^−/− cells (8 and 4a) are viable as determined by trypan blue exclusion assays. Each cell viability assay was performed in duplicate, and the results shown represent the mean values ± SEM of three independent experiments, presented as the percentage of viable cells. In addition, replated N-Adh Tsc2^−/− cells grow in foci-like colonies (right panels). (E) N-Adh Tsc2^−/− cells undergo multiple rounds of cell proliferation as determined by BrdU-Hoechst flow cytometry. At confluence, BrdU was added to the media for 72 hours. Nonadherent cells were collected and processed for analyses. Plots depict the number of cells versus Hoechst intensity and show the distribution of cells in G0/G1, G1′, and G1" phases representing the relative proportion of cells in the first, second, and third cell cycle postlabeling, respectively.

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