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Fig. 2 | Genome Medicine

Fig. 2

From: A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene

Fig. 2

The isoform with the p.L605F variant impairs FANCI stability and function. a Western blots of HeLa cells with the FANCI gene (FANCI+/+) or with the FANCI gene knocked out (FANCI−/−). HeLa FANCI−/− cells from clone 1 were complemented with constructs of Flag-FANCI wild type (WT), p.L605F or p.P55L, or an empty vector (EV) and treated with 50 ng/ml MMC for 18 h. The upper band, H, shows the ubiquitination of FANCI and FANCD2 after treatment. The lower band, L, corresponds to non-ubiquitinated FANCI or FANCD2. VINCULIN was used as a loading control. Experiment was repeated three times. b HeLa FANCI+/+ cells were transfected with siRNA targeting FANCI and then complemented with Flag-FANCI siRNA-resistant constructs or an EV. Cells were treated with 50 ng/ml MMC for 18 h followed by FLAG immunoprecipitation. The left panel shows FANCI constructs expression and the right panel the immunoprecipitated fractions. The p.L605F immunoprecipitation fraction sample was super-loaded to have the same signal after FANCI WT complementation. The ratio between the upper band (H) and lower band (L) for the immunoprecipitated FANCD2 is shown. c Immunofluorescence of HeLa FANCI−/− cells from clone 1 that were complemented with constructs of Flag-FANCI and 0.1 μg of empty GFP vector was used as a transfection control. The adjacent scatter plot shows the number of FANCD2 foci in GFP-positive cells after treatment with MMC (50 ng/ml, 18 h). Mean with SEM is represented. The Kruskal-Wallis test was used to compare groups and the P value is shown for each test. Experiment has been performed in triplicate. d–f Western blot analysis of HeLa FANCI−/− cells from clone 1 that were complemented with constructs of Flag-FANCI and treated with cycloheximide (CHX) and either mock-treated (d) or treated with damaging agents formaldehyde (e) or MMC (f) for different lengths of time at the indicated concentrations. At each time point, whole cell extracts were analysed by western blot to assess protein levels. Experiment has been done in triplicate. g Survival curves of HeLa FANCI−/− cells from clone 1 that were transfected with the different constructs of Flag-FANCI. Cell viability was monitored following cisplatin or olaparib treatments for 72 h and was assessed by counting remaining nuclei. Curves represent mean with SEM of three biological replicates. Western blots were used to monitor expression and shown here as an example. Alpha-tubulin was used as a loading control. Full blots are shown in Additional file 4

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