Journal: Scientific Reports

Article Title: Coumarin-chalcone hybrid instigates DNA damage by minor groove binding and stabilizes p53 through post translational modifications

doi: 10.1038/srep45287

Figure Lengend Snippet: ( A ) HCT116 cells were treated with 7.5 μM of S009-131 for indicated time points. Whole cell lysates were then collected and analysed by immunoblotting using antibodies specific to ATM, ATR and DNA-PK and their active phosphorylated form. ( B–D ) Cells were incubated with S009-131 with or without pre-treatment with specific PIKK inhibitors (KU5993, 10 μM; NU7441, 10 μM and VE821, 5 μM) and analysed for γ-H2AX expression by immunoblotting.

Article Snippet: NU7441, KU55933, rabbit anti-p53 (sc-6243, Lot #A1212), mouse anti-p53 (sc-126, Lot #F1113), mouse anti-MDM2 (sc-965, Lot #E0813), mouse anti-ATM (sc-23921, Lot #F2113), goat anti-ATR (sc-1887, Lot #E1613), mouse anti-DNA-PKcs (sc-5282, Lot #H0613) and mouse anti-p21 (sc-6246, Lot #I0111) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, California, USA).

Techniques: Western Blot, Incubation, Expressing

Journal: Scientific Reports

Article Title: Coumarin-chalcone hybrid instigates DNA damage by minor groove binding and stabilizes p53 through post translational modifications

doi: 10.1038/srep45287

Figure Lengend Snippet: ( A ) HCT116 cells were treated with S009-131 for different time points. The protein lysates were then analysed by immunoblotting to detect phosphorylation state of p53 at indicated residues. ( B–D ) Influence of PIKKs on S009-131 induced p53 phosphorylation at Ser 15 was assessed by 2 h pre-incubation of cells with caffeine (1 mM), Wortmannin (10 μM), LY29002 (20 μM) and ( E–G ) with specific inhibitors.

Article Snippet: NU7441, KU55933, rabbit anti-p53 (sc-6243, Lot #A1212), mouse anti-p53 (sc-126, Lot #F1113), mouse anti-MDM2 (sc-965, Lot #E0813), mouse anti-ATM (sc-23921, Lot #F2113), goat anti-ATR (sc-1887, Lot #E1613), mouse anti-DNA-PKcs (sc-5282, Lot #H0613) and mouse anti-p21 (sc-6246, Lot #I0111) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, California, USA).

Techniques: Western Blot, Phospho-proteomics, Incubation

Journal: Scientific Reports

Article Title: Coumarin-chalcone hybrid instigates DNA damage by minor groove binding and stabilizes p53 through post translational modifications

doi: 10.1038/srep45287

Figure Lengend Snippet: ( A ) HCT116 cells were co-incubated with 10 μM MG132 and 7.5 μM S009-131. Whole cell lysates were analysed by immunoblotting with anti-p53 antibody. ( B ) Cell lysates were also subjected to immunoprecipitation (IP) using 1 μg of anti-p53 antibody followed by Western blot for p53 associated ubiquitins. ( C ) Predicted binding models for S009-131 and chalcone with MDM2. ( D ) HCT116 cells were treated with S009-131 and Nutlin at indicated concentrations for 24 h. Five hundred micrograms of protein lysates were immunoprecipitated with anti-MDM2 antibody and bound p53 was detected by immunoblotting. ( E and F ) Band intensities of Co-IP assay were analysed by Image J software and plotted graphically. ( G ) Western blot assay was performed to show the effect of S009-131 (10 μM) on p53-MDM2 interaction after immuonoprecipitating protein lysates with anti-p53 antibody. ( H and I ) Band intensities were determined by Image J software and represented graphically. ( J ) Binding affinity of p53 towards responsive elements (p53-RE) at promoter region before and after S009-131 treatment was determined by co-transfecting cells with EGFP-C1 and p53-Luc Cis Reporter plasmids. Luminescence were normalised by comparing with relative fluorescence in treated and untreated cells. ( K ) Western blot assay of whole cell lysates from S009-131 treated HCT116 cells to determine p21 protein level. ( L ) Chromatin immunoprecipitation (ChIP) assay was performed by immunoprecipitating chromatin complex from S009-131 treated and untreated cells with anti-p53 antibody followed by quantitative RT-PCR using p21 promoter specific primers.

Article Snippet: NU7441, KU55933, rabbit anti-p53 (sc-6243, Lot #A1212), mouse anti-p53 (sc-126, Lot #F1113), mouse anti-MDM2 (sc-965, Lot #E0813), mouse anti-ATM (sc-23921, Lot #F2113), goat anti-ATR (sc-1887, Lot #E1613), mouse anti-DNA-PKcs (sc-5282, Lot #H0613) and mouse anti-p21 (sc-6246, Lot #I0111) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, California, USA).

Techniques: Incubation, Western Blot, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay, Software, Fluorescence, Chromatin Immunoprecipitation, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Reduced recruitment of 53BP1 during interstrand crosslink repair is associated with genetically inherited attenuation of mitomycin C sensitivity in a family with Fanconi anemia

doi: 10.18632/oncotarget.23375

Figure Lengend Snippet: ( A–D ) DDR analyses in SV40-transformed fibroblasts from a healthy control, the mother, the ATM R32C/WT clone #1, the ATM R32C/- clone #14, and in AT5BIVA cell line. (A) Western blot analysis of the IR-induced phosphorylation of ATM at S1981 and Kap1 at S824 one hour after 10 Gy irradiation. (B) Representative images showing phospho-ATM S1981 IR-induced foci (green staining) one hour after 10 Gy irradiation. DAPI: blue staining. (C) Percentages of G 2 cells entering mitosis following 0.5 or 5 Gy irradiation calculated related to untreated. Results are presented as mean ± SD of 3 independent experiments. (D) Cell survival after exposure to increasing doses of IR. Results shown as mean ± SD of duplicates are representative of 3 independent experiments.

Article Snippet: Whole cell lysates were analyzed by SDS-PAGE and blotted with the following antibodies: polyclonal rabbit anti-FANCD2 (Santa Cruz sc-28194); monoclonal mouse anti-ATM (clone 2C1, Santa Cruz sc-23921); monoclonal rabbit anti-phospho-ATM S1981 (Novus NB110-66655); polyclonal rabbit anti-phospho-Kap1 S824 (Bethyl Laboratories IHC-00073) and mouse monoclonal anti-Ku70 (Santa Cruz sc-17789).

Techniques: Transformation Assay, Control, Western Blot, Phospho-proteomics, Irradiation, Staining