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Fig. 1 | Orphanet Journal of Rare Diseases

Fig. 1

From: High content drug screening for Fanconi anemia therapeutics

Fig. 1

FA cell-based system setup. a Upon DNA damage causing ICLs on DNA, FA/BRCA pathway is activated, FANCD2 ubiquitinated, and fluorescent FANCD2 foci can be seen on the microscope (left). Deficient FA/BRCA pathway is not able to ubiquitinate FANCD2 and no FANCD2 foci can be detected (middle). A drug able to overcome FANCA deficiency on FA/BRCA pathway can be addressed with this cell-based system. FANCD2 foci can be clearly identified from the nucleus FANCD2 background signal (right). b Lack of FANCA expression of two positive clones after TALEN-mediated gene targeting. Cells were treated with MMC at 10 and 33 nM for 24 h, lyzed and FANCA expression and FANCD2 ubiquitination analyzed by Western blot. Vinculin was used as a loading control. c Survival assay of FANCA-deficient U2OS clones. 2F8 and 2G7 clones and WT cells were treated with a MMC dose curve from 1 to 100 nM. 72 h later cells were collected and counted. Graph shows mean survival percentage respect non-treated cells +/− SEM of at least three independent experiments with similar results. d 2F8 FANCA-deficient U2OS clone was stably transfected with YFP-FANCD2 (see materials and methods). FANCA-deficient cells (left lanes) and FANCA corrected cells (right lanes) were treated with 2 mM HU for 24 h, then lyzed and FANCA, endogenous FANCD2 and exogenous YFP-FANCD2 expression analyzed by Western blot. Vinculin was used as a loading control. e Fluorescence (green) of YFP-FANCD2 stably transfected on WT (left), FANCA-deficient (middle), and FANCA corrected (right) U2OS cell lines was analyzed by microscopy after treatment with 2 mM HU for 24 h. Bottom images show overlay of YFP-FANCD2 fluorescence with DAPI (nuclei staining)

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