Research Paper Volume 12, Issue 14 pp 14791—14807

Figure 2. MiR-185 induces DNA damage foci at dysfunctional telomeres by decreasing POT1 protein level in cancer cells. (A) POT1 mRNA levels in POT1 knockdown, miR-185 overexpressing and POT1 rescue HTC75 cells. (B) miR-185 levels in POT1 knockdown, miR-185 overexpression and POT1 rescuing HTC75 cells. (C) POT1 protein level in POT1 knocking-down, miR-185 overexpression and POT1 rescue HTC75 cells. The arrowhead indicates exogenously overexpressed POT1. GAPDH was blotted as a loading control. (D) Immunofluorescence and fluorescence in situ hybridization (IF-FISH) were performed in POT1 knockdown, miR-185 overexpression and POT1 rescue HTC75 cells with the indicated γ-H2AX antibody and the TTAGGG telomere probe. Nuclei were stained with Hoechst 33342. (E) Quantification of percentage of cells in (D) with more than 3 TIFs. Error bars indicate standard deviations (n=3). P values were determined by Student’s t-test. ***P<0.001. (F) Quantification of the percentage of cells with signal indicating total DNA damage (γ-H2AX positive cells) in (D).