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ECs are defective in HR repair.

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posted on 12.12.2012, 02:01 by Francesca Cavallo, Grazia Graziani, Cristina Antinozzi, Darren R. Feldman, Jane Houldsworth, George J. Bosl, Raju S. K. Chaganti, Mary Ellen Moynahan, Maria Jasin, Marco Barchi

A) EC cells are defective in RAD51 foci assembly. The indicated cell lines were treated with a pulse of cisplatin (3.3 µM for 6 hs) and co-stained with anti-RAD51 (red) and anti-bromodeoxyuridine (BrdU, [green]) antibodies. Harrows points to representative BrdU-positive (S-phase) cells used for RAD51 quantification (see below). B) cisplatin induces a comparable damage in U2OS and EC cell lines. The indicated cell lines were treated as in A, co-stained with γH2AX (red) and BrdU (green) antibodies, and counterstained with DAPI (blue). Harrows points to representative BrdU-positive (S-phase) cells used for γH2AX quantification. C–D) quantification of the number of RAD51 (C) and γH2AX (D) foci, before and after cisplatin treatment. Data are mean value ±s.d. of two independent experiments. In C and D a minimum of 100 nuclei were counted for each cell line. Statistical analysis was performed using a paired two-tailed Student's t-test (P<0.05). E) 27x-1 and Tera-1 cell lines are defective in I-SceI-induced DSB,repair, by HR. Percentage of GFP+ cells measured by flow cytometry, 48 hs upon the transfection with both DR-GFP and I-SceI expression vectors (black bars, [DR-GFP+I-SceI]). Data were normalized against the transfection efficiency measured by transfecting a (constitutive) GFP-expressing vector (white bars [NZE CAG]). Data are mean value ± s.d. of three independent experiments. Statistical analysis was performed using a paired two-tail Student’s t-test (P<0.05). For more details see also Fig. S3A–B.

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