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Fig. 3 | BMC Cancer

Fig. 3

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

Fig. 3

CRL4 deficiency impairs the interstrand crosslinks dependent assembling of ssDNA-RPA complexes. a HeLa cells were transfected with the indicated siRNA and labeled with IdU prior to cisplatin exposure for 24 h. After fixation, cells were stained for RPA2 and ssDNA. DAPI was used to visualize the nuclei. b Quantification of nuclear fluorescence representing RPA2 foci (N = 360–680 nuclei from 3 to 6 experiments). c Quantification of nuclear fluorescence representing ssDNA induced by the indicated treatments (N = 230–530 nuclei from 3 to 4 experiments). d HeLa cells were transfected with siRNA and labeled with IdU prior to MMC exposure for 24 h. For the detection of pS33 (RPA2 phosphorylated at Ser33), fixed cells were stained with phospho-specific antibodies. DAPI was used to visualize the nuclei. e SKOV3 cells were transfected with siRNA as indicated, treated for 24 h with MMC, and stained for pS33 and ssDNA. f Quantification of nuclear fluorescence representing ssDNA and pS33 foci in HeLa cells (N = 262–349 nuclei from 2 experiments). g Quantification of ssDNA and pS33 foci in SKOV3 cells (N = 200–240 nuclei from 2 experiments). In panels b, c, f, g, horizontal lines represent median values, boxes 25-75th percentiles and whiskers 10-90th percentiles. *P < 0.05, ***P < 0.001 (1-way ANOVA according to Kruskal-Wallis). Scale bar: 10 μm

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