By the dark DAPI staining and constrictions of sister chromatids.Immunofluorescence

By the dark DAPI staining and constrictions of sister chromatids.Immunofluorescence StainingImmunofluorescence staining was performed as described [46]. Primary antibodies against centromere antigens (centromere autoantibodies) (The Binding Site, Birmingham, UK) and cH2AX (Upstate, Lake Placid, NY, USA) were applied at a dilution of 1:500. Suitable secondary antibodies conjugated with AlexaFluor 488 or rhodamine (Molecular Probes, Eugene, OR, USA) were used for dual-color staining. Cells were counterstained with 49,6-diamino-2-phenylindole (DAPI, Applied Spectral Imaging, Vista, CA, USA). Immunofluorescence images were captured using a confocal laser scanning microscope (Zeiss LSM 510, Jena, Germany).Western Blotting and Flow Cytometric AnalysisWestern Blotting and flow cytometric analysis of cell cycle were performed as previously reported [32]. For Western Blotting, fifteen microgram protein was separated by SDS-PAGE and blots were prepared on a polyvinylidene fluoride membrane (Amersham, Piscataway, NJ, USA). Primary antibodies against p16INK4a and actin were from NeoMarkers (Fremont, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The membrane was probed with secondary antibody against KS 176 biological activity peroxi-Statistical AnalysisThe two-tailed T-test was performed to analyze the statistical differences. P values ,0.05 were considered as statistically significant. In all bar graphs, error bars represent standard deviations.Centromeric Instability after Replication StressSupporting InformationFigure S1 Typical SKY karyotypes at late passages of(TIF)Table S1 Structural chromosome aberrations in esophageal and cervical epithelial cells expressing HPV16 E6E7 and hTERTa. (DOC) Table S2 Karyotype at population doubling 80 of cell lines immortalized by HPV16 E6E7 and hTERT a. (DOC)two immortalized esophageal epithelial cell lines expressing HPV16 E6E7 and hTERT. Arrows indicate chromosomes with centromeric or pericentromeric aberrations. (TIF)Figure S2 Typical SKY karyotypes at late passages oftwo immortalized cervical epithelial cell lines expressing HPV16 E6E7 and hTERT. Arrows indicate chromosomes with centromeric or pericentromeric aberrations. (TIF)Figure S3 Western Blotting for p16INK4a. Actin bandsAcknowledgmentsWe thank Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, for use of SKY facilities. We also thank T. Chan, C. S. Leung, P. Mak, J. Cheung, A. Li for technical assistance.served as protein load controls. NC104 cells at PD 18 were approaching permanent growth arrest (PD20), which was included to show up-regulation of p16 INK4a for the comparison with p16 INK4a levels after HPV 16 E6E7 4EGI-1 site expression. (TIF)Figure S4 Flow cytometric analysis of cell cycle distributions. Only the quantitative data for percentages of Sphases were given for simplicity.Author ContributionsConceived and designed the experiments: WD ALMC. Performed the experiments: WD. Analyzed the data: WD ALMC. Contributed reagents/ materials/analysis tools: SWT XYG. Wrote the paper: WD ALMC.
The Gram-negative bacterium Vibrio cholerae is the causative agent of the acute diarrheal disease cholera and remains a serious health risk to humans. In addition to the two main virulence factors needed to cause massive watery diarrhea holera toxin [1] and the toxin coregulated pilus [2] he bacterium utilizes accessory virulence factors also capable of causing diarrheal disease. Accessory toxins such as hemolysin (HlyA) and actin-crosslinkin.By the dark DAPI staining and constrictions of sister chromatids.Immunofluorescence StainingImmunofluorescence staining was performed as described [46]. Primary antibodies against centromere antigens (centromere autoantibodies) (The Binding Site, Birmingham, UK) and cH2AX (Upstate, Lake Placid, NY, USA) were applied at a dilution of 1:500. Suitable secondary antibodies conjugated with AlexaFluor 488 or rhodamine (Molecular Probes, Eugene, OR, USA) were used for dual-color staining. Cells were counterstained with 49,6-diamino-2-phenylindole (DAPI, Applied Spectral Imaging, Vista, CA, USA). Immunofluorescence images were captured using a confocal laser scanning microscope (Zeiss LSM 510, Jena, Germany).Western Blotting and Flow Cytometric AnalysisWestern Blotting and flow cytometric analysis of cell cycle were performed as previously reported [32]. For Western Blotting, fifteen microgram protein was separated by SDS-PAGE and blots were prepared on a polyvinylidene fluoride membrane (Amersham, Piscataway, NJ, USA). Primary antibodies against p16INK4a and actin were from NeoMarkers (Fremont, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The membrane was probed with secondary antibody against peroxi-Statistical AnalysisThe two-tailed T-test was performed to analyze the statistical differences. P values ,0.05 were considered as statistically significant. In all bar graphs, error bars represent standard deviations.Centromeric Instability after Replication StressSupporting InformationFigure S1 Typical SKY karyotypes at late passages of(TIF)Table S1 Structural chromosome aberrations in esophageal and cervical epithelial cells expressing HPV16 E6E7 and hTERTa. (DOC) Table S2 Karyotype at population doubling 80 of cell lines immortalized by HPV16 E6E7 and hTERT a. (DOC)two immortalized esophageal epithelial cell lines expressing HPV16 E6E7 and hTERT. Arrows indicate chromosomes with centromeric or pericentromeric aberrations. (TIF)Figure S2 Typical SKY karyotypes at late passages oftwo immortalized cervical epithelial cell lines expressing HPV16 E6E7 and hTERT. Arrows indicate chromosomes with centromeric or pericentromeric aberrations. (TIF)Figure S3 Western Blotting for p16INK4a. Actin bandsAcknowledgmentsWe thank Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, for use of SKY facilities. We also thank T. Chan, C. S. Leung, P. Mak, J. Cheung, A. Li for technical assistance.served as protein load controls. NC104 cells at PD 18 were approaching permanent growth arrest (PD20), which was included to show up-regulation of p16 INK4a for the comparison with p16 INK4a levels after HPV 16 E6E7 expression. (TIF)Figure S4 Flow cytometric analysis of cell cycle distributions. Only the quantitative data for percentages of Sphases were given for simplicity.Author ContributionsConceived and designed the experiments: WD ALMC. Performed the experiments: WD. Analyzed the data: WD ALMC. Contributed reagents/ materials/analysis tools: SWT XYG. Wrote the paper: WD ALMC.
The Gram-negative bacterium Vibrio cholerae is the causative agent of the acute diarrheal disease cholera and remains a serious health risk to humans. In addition to the two main virulence factors needed to cause massive watery diarrhea holera toxin [1] and the toxin coregulated pilus [2] he bacterium utilizes accessory virulence factors also capable of causing diarrheal disease. Accessory toxins such as hemolysin (HlyA) and actin-crosslinkin.