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. 2018 Jun 15;7(7):bio034181. doi: 10.1242/bio.034181

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© 2018. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — hTERT defines CSC properties. (A) Confocal immunofluorescence images for ALDH1A1 (green) showing that hTERT^high CSCs are positive for ALDH1A1, whereas hTERT^-/low CSCs do not express ALDH1A1. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Confocal immunofluorescence images for β-catenin (green) showing cytoplasmic localization of β-catenin in hTERT^-/low CSCs, whereas hTERT^high CSCs showed nuclear localization of β-catenin. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (C) Side population (SP) analysis by flow cytometry indicating more SP cells in hTERT^high CSCs than in hTERT^-/low CSCs, which have fewer SP cells than do control CSCs. (D) Flow cytometry analysis of CD133 showing that hTERT^high CSCs have higher CD133 expression than do control CSCs, whereas hTERT^-/low CSCs are negative for CD133 expression. An isotype control was used to define the positive and negative populations. (E–F) hTERT^high CSCs, hTERT^-/low CSCs and control CSCs were exposed to increasing concentrations of cisplatin (E) or doxorubicin (F) for 24 h. Cell viability was determined by Annexin-V-FITC and PI apoptosis detection kits. hTERT^high CSCs showed more significant resistance to cisplatin and doxorubicin than did control CSCs, whereas hTERT^-/low CSCs exhibited a relative loss of chemoresistance capabilities (P<0.05). (G) The expression levels of the cancerous markers E2F3, HER2, KRas, SMAD7, TP53, CDK4 and CDK6 are higher in hTERT^high CSCs as determined by real-time qRT-PCR. The data are reported as the means±s.d. (H) Real-time qRT-PCR analysis of CSC marker genes showing higher expression levels in hTERT^high CSCs. β-actin mRNA was used to normalize variability in template loading. The data are reported as the means±s.d. (I) Real-time qRT-PCR analysis of Bcl-2 (an anti-apoptotic protein) and Bax (a pro-apoptotic molecule indicating a significantly increased expression of Bcl-2 and reduced expression of Bax in hTERT^high CSCs compared with that in control CSCs (P<0.05). β-actin mRNA was used to normalize the variability in template loading. The data are reported as the means±s.d.