Journal: NPJ Breast Cancer
Article Title: The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer
Figure Lengend Snippet: Functional studies of the FANCM :p.Arg658*, p.Gln1701* and p.Arg1931* truncating variants using the patient-derived FANCM − / − EGF280 cell line. a Western blot showing the FANCM expression in EGF280 cells complemented with lentiviral vectors harboring the three different variants. Bands corresponding to truncated FANCM protein were visible for EGF280 + p.Gln1701* and p.Arg1931*, and no bands were present for the EGF280 + p.Arg658*. b Study of the expression of the FANCM protein in EGF280 + p.Arg658*. The c.1972C > T base substitution, causing the p.Arg658* variant abrogates a digestion site for the restriction enzyme Tse I that is present in the wild-type (wt) cDNA sequence. Total RNA was extracted from EGF280 + wt FANCM and from the EGF280 + p.Arg658* and subjected to reverse transcription. PCR-amplified cDNA products were digested with Tse I. Digested and undigested cDNAs were loaded. In the first two lanes are shown bands of 386 bp corresponding to uncut wt cDNA, and bands of 257 and 129 bp corresponding to cut wt cDNA. In next two lanes bands of 386 bp indicate that p.Arg658* cDNA was not cut due to the c.1972C > T base substitution abrogating the Tse I site. In the two lanes after the molecular weight marker (M) undigested and digested products of the two previous PCR products were mixed 1:1 and loaded as a control. c Analysis of diepoxybutane (DEB) sensitivity on cell survival. The EGF280 cells expressing p.Arg658* are significantly more sensitive to DEB than the cells expressing p.Gln1701* or p.Arg1931* ( P-values from Tukey’s range test are reported in Supplementary Table 4 ). EGF280 and EGF280 + wtFANCM are used as controls ( N = 3; error bars: standard deviation). d Chromosome fragility induced by DEB treatment (100 ng/ml). Here, the chromatidic break patterns of the cells expressing wt FANCM, of the cells harboring p.Gln1701* or p.Arg1931* variants, and of the native EGF280 cells or the cells expressing p.Arg658* were statistically different. ( P-values from chi-squared test; N = 2). e Analysis of cellular sensitivity to olaparib. Contrarily to what we observed in the DEB sensitivity assays, survival rates of the different complemented cell lines were apparently not different. Human fibroblasts ( BRCA2 − / − ) were homozygous for the c.469 A > T (p.Lys157*) truncating variant and were used as a positive control. ( P-values from Tukey’s range test are reported in Supplementary Table 5 ; N = 3; error bars: standard deviation). All blots derive from the same experiment and were processed in parallel
Article Snippet: Reverse transcription was performed using High-Capacity RNA-to-cDNA Kit (Thermofisher); a cDNA region corresponding to the FANCM sequence containing the amino acid (AA) position Arg658 was amplified by PCR using the forward: 5’-AGTAACAGGCAGGTCCTTCA-3´and reverse: 5’-TGATCTTGCCACAGTCTCCA-3’ primers.
Techniques: Functional Assay, Derivative Assay, Western Blot, Expressing, Variant Assay, Sequencing, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Standard Deviation, Positive Control