Figure 4.
Figure 4. Limiting dilution culture of lung cells induced to express KRASG12D in vitro. (A) Phase-contrast images of AdCMVCre-infected KRASLSL-G12D lung cell cultures (2 × 105 per well plating). Cultures developing floating cell clusters composed of small round cells (left), macrophage-like large round cells with fibroblasts (middle), and poorly growing cells (right), are indicated. Original magnification ×200. (B) Comparison between AdCMVCre-infected and control (AdCMVβgal or AdSPCCre-infected) cultures with regard to the development of floating round cell clusters and macrophage-like cells. A total of 20 wells plated at 2 × 105 per well (5 wells per mouse × 4 KRASLSL-G12D mice) for each infection were analyzed at 4 weeks in culture. Floating round cell cluster development was detected only in AdCMVCre-infected cultures (3 of 20 wells). Statistical significance of the difference was confirmed by χ2 test. (C) Limiting dilution analysis to determine the frequency of the cells initiating the development of floating round cell clusters. The data obtained from adenovirus-infected cultures plated at 2 × 105 per well (20 wells), 5 × 105 per well (12 wells), or 1 × 106 per well (8 wells), and floating cell cluster formation was determined at 4 weeks in culture. The frequency was estimated as 1 in 0.83 × 106 AdCMVCre-infected cells. (D) PCR detection of recombination of the KRASG12D allele in AdCMVCre or AdCMVβgal-infected cultures (top), and in small round cells forming floating cell clusters or macrophage-like large cells obtained from AdCMVCre-infected cultures (bottom). The recombined KRASG12D allele (Lox-G12D) was detected in both floating small cells and large macrophage-like cells developing in the AdCre-infected culture, at a similar level to the KRASWT (WT) allele. (E) Flow cytometry analysis of surface CD11c and F4/80 expression on small cells forming floating clusters at 7 weeks in the AdCMVCre-infected culture. A representative contour plot for CD11c and F4/80 is indicated. (F) Representative phase-contrast (top, original magnification ×200) and CD207/MAC2 immunofluorescence (bottom, by confocal laser scanning microscopy) images of replated cluster cells. Floating cells at 7 weeks in the AdCMVCre-infected culture were replated on coverslips, and imaged at 48 hours following replating. Scale bar, 20 μm (confocal images).

Limiting dilution culture of lung cells induced to express KRASG12Din vitro. (A) Phase-contrast images of AdCMVCre-infected KRASLSL-G12D lung cell cultures (2 × 105 per well plating). Cultures developing floating cell clusters composed of small round cells (left), macrophage-like large round cells with fibroblasts (middle), and poorly growing cells (right), are indicated. Original magnification ×200. (B) Comparison between AdCMVCre-infected and control (AdCMVβgal or AdSPCCre-infected) cultures with regard to the development of floating round cell clusters and macrophage-like cells. A total of 20 wells plated at 2 × 105 per well (5 wells per mouse × 4 KRASLSL-G12D mice) for each infection were analyzed at 4 weeks in culture. Floating round cell cluster development was detected only in AdCMVCre-infected cultures (3 of 20 wells). Statistical significance of the difference was confirmed by χ2 test. (C) Limiting dilution analysis to determine the frequency of the cells initiating the development of floating round cell clusters. The data obtained from adenovirus-infected cultures plated at 2 × 105 per well (20 wells), 5 × 105 per well (12 wells), or 1 × 106 per well (8 wells), and floating cell cluster formation was determined at 4 weeks in culture. The frequency was estimated as 1 in 0.83 × 106 AdCMVCre-infected cells. (D) PCR detection of recombination of the KRASG12D allele in AdCMVCre or AdCMVβgal-infected cultures (top), and in small round cells forming floating cell clusters or macrophage-like large cells obtained from AdCMVCre-infected cultures (bottom). The recombined KRASG12D allele (Lox-G12D) was detected in both floating small cells and large macrophage-like cells developing in the AdCre-infected culture, at a similar level to the KRASWT (WT) allele. (E) Flow cytometry analysis of surface CD11c and F4/80 expression on small cells forming floating clusters at 7 weeks in the AdCMVCre-infected culture. A representative contour plot for CD11c and F4/80 is indicated. (F) Representative phase-contrast (top, original magnification ×200) and CD207/MAC2 immunofluorescence (bottom, by confocal laser scanning microscopy) images of replated cluster cells. Floating cells at 7 weeks in the AdCMVCre-infected culture were replated on coverslips, and imaged at 48 hours following replating. Scale bar, 20 μm (confocal images).

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