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Fig. 5 | BMC Cancer

Fig. 5

From: CRL4 ubiquitin ligase stimulates Fanconi anemia pathway-induced single-stranded DNA-RPA signaling

Fig. 5

CRL4 depletion impairs recruitment of FANCD2 and XPF-ERCC1. a HeLa cells were transfected with siRNA and labeled with IdU prior to 24-h cisplatin exposures. After fixation, cells were stained for FANCD2 and ssDNA (using an anti-IdU antibody). DAPI was used to visualize the nuclei. b Quantification of nuclear fluorescence representing FANCD2 foci induced as indicated (N = 590–700 nuclei from 3 experiments). c Cells were transfected with siRNA and labeled with IdU prior to 24-h cisplatin exposures. After fixation, cells were stained for ERCC1 and ssDNA. DAPI was used to visualize the nuclei. d Quantification of nuclear fluorescence representing ERCC1 foci (N = 350–480 nuclei from 3 experiments). e Immunoblot showing the monoubiquitination of FANCD2 after 24-h cisplatin exposures. Upper bands represent monoubiquitinated FANCD2. Tubulin was used as the loading control. f Ratio of monoubiquitinated FANCD2 (FANCD2-Ub) and unmodified FANCD2 (N = 3). In panels b and d, horizontal lines represent median values, boxes the 25-75th percentiles and whiskers the 10-90th percentiles. ***P < 0.001 (1-way ANOVA according to Kruskal-Wallis). In panel f, data are presented as mean ± SEM. *P ≤ 0.05 (unpaired, two-tailed t-test). Scale bar: 10 μm

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