Journal: Radiation Oncology (London, England)

Article Title: IL-6 signaling promotes DNA repair and prevents apoptosis in CD133+ stem-like cells of lung cancer after radiation

doi: 10.1186/s13014-015-0534-1

Figure Lengend Snippet: IL-6 knocked down CD133+ cells showed lower expression of DNA repair-associated molecules. a Higher expression of γ-H2AX indicating unrepaired DNA damage was found in IL-6 knocked down CD133+ cell cultures measured by IF staining. CD133+ cells of A549IL-6si/sc and H157IL-6si/sc pairs were irradiated with 6 Gy gamma rays and γ-H2AX IF staining was performed at 3 h after irradiation. b Higher DNA strand breaks were detected in CD133+ cells of IL-6si cells than those of sc cells. CD133+ and CD133- cells were obtained from A549IL-6si/sc and H157IL-6si/sc cell pairs, irradiated (6 Gy), and DNA strand breaks were analyzed at 0 and 30 min after radiation in Comet assay. The ratio of head diameter to comet length of 50 cells were measured in each sample analysis and used in quantitation shown on right. c Lower expression of DNA repair associated proteins, ATM, CHK2, p-ATM, and p-p53 were detected in CD133+ cells of IL-6 knocked down cell line than those of sc counterparts (IF staining). CD133+ and CD133- cells of A549IL-6si/sc and H157IL-6si/sc pairs were irradiated with 6 Gy gamma rays and IF staining was performed at 2 h after irradiation using antibodies of ATM, CHK2, p-ATM, and p-p53. d qPCR analysis. CD133+ cells of A549IL-6si/sc and H157IL-6si/sc pairs were irradiated with 6 Gy gamma rays and total RNAs were obtained for qPCR analyses at 6 h after irradiation. e ATM-luc assay. 293HEK and H1299 cells were transfected with ATM-luc and cultured with various concentrations of IL-6. At the end of 48 h of incubation, luciferase activities were measured

Article Snippet: Antibodies of ATM and CHK2 were obtained from Bethyl Laboratory (Montgomery, TX), phosphorylated ATM (Ser 1981), and phosphorylated p53 (Ser 20) were from Gene Tex (Irvine CA), and γ-H2AX antibody was purchased from Trevigen (Gaithersburg, MD).

Techniques: Expressing, Staining, Irradiation, Single Cell Gel Electrophoresis, Quantitation Assay, Transfection, Cell Culture, Incubation, Luciferase

Journal: The Journal of Neuroscience

Article Title: ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

doi: 10.1523/JNEUROSCI.2635-12.2013

Figure Lengend Snippet: Axotomized RGCs die in a p53-dependent manner. A, RT-PCR analysis revealed that p53 mRNA levels do not change at 1 or 3 d (shown here) after axotomy with respect to noninjured retinas (Student's t test, p > 0.05). B, In contrast, a significant increase in axotomy-induced phosphorylation of p53 at serine 15 (S15) was readily detected at 1 d after axotomy, but returned to basal levels at 7 d postlesion (ANOVA, *p < 0.05). C, Retinal immunostaining of Fluorogold-labeled RGCs confirmed that p53 phosphorylation (activation) was detected in these neurons at 1 d after axotomy. Scale bars, 10 μm. D, Protein levels of the p53 apoptotic targets PUMA and Fas/CD95 increased at 1 d after axotomy, but returned to normal levels at 7 d postinjury. Bax and Noxa remained unchanged. E, Analysis of RGC loss quantified at 1 week after axotomy in p53-null, heterozygote, and wild-type retinas demonstrated an allelic dose dependency on p53 (ANOVA, ***p < 0.001). Data are expressed as RGC densities (RGCs/mm2; mean ± SD).

Article Snippet: Phosphorylation of p53 at S15 has been shown to be sufficient to induce apoptosis of human glioma cells and leads to selective increase of the proapoptotic p53 targets Fas/CD95 and PUMA ( Amano et al., 2009 ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Immunostaining, Labeling, Activation Assay