Additional file 1 of Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays
Creators: name: Bassi, Nicola
name: Hovland, Henrikke Nilsen
name: Rasheed, Kashif
name: Jarhelle, Elisabeth
name: Pedersen, Nikara
name: Mchaina, Eunice Kabanyana
name: Bakkan, Sara Marie Engelsvold
name: Iversen, Nina
name: Høberg-Vetti, Hildegunn
name: Haukanes, Bjørn Ivar
name: Knappskog, Per Morten
name: Aukrust, Ingvild
name: Ognedal, Elisabet
name: Van Ghelue, Marijke
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1:Supplementary Table 1. Primer sequences used in site-directed mutagenesis. Supplementary Figure 1. Protein expression levels of BRCA1 variants determined by western blot analysis. (A) The figure shows images from one representative replicate for the fusion protein DBD-BRCT. HEK293FT cells were transiently transfected with DBD-BRCT WT, known benign (green) and pathogenic (red) control variants, and 11 missense BRCA1 VUSs. Cells were harvested 48 h post transfection, and 7 µg total protein retrieved from cell lysate was analysed per lane by western blotting. BRCA1 was detected with anti-BRCA1 antibody. The BRCA1 bands were normalised against the total protein of each lane. The blots were cut in two prior to incubation with anti-BRCA1 and anti-actin, so each blot are divided into two images. (B) The figure shows images from one representative replicate for the full-length His-BRCA1. HEK293FT cells were transiently transfected with His-BRCA1 WT, known benign (green) and pathogenic (red) control variants, and 11 missense BRCA1 VUSs. Cells were harvested 48 h post transfection, and 5 µg total protein retrieved from cell lysate was analysed per lane by western blotting. BRCA1 was detected with anti-BRCA1 antibody. Actin was used as loading control to normalise the corresponding BRCA1 bands. Supplementary Figure 2. Representative raw data output from flow cytometry. The settings for analyses were: FSC=60, SSC=220, GFP=235. Footnotes: FSC, Forward SCatter; SSC, Side SCatter; GFP, Green Fluorescent Protein; WT, Wild Type; I-SceI, endonuclease causing double strand break; sc siRNA, scrambled siRNA. Supplementary Figure 3. Fluorescent microscope analyses of GFP+ cells after HRR induction. The cells were evaluated 24 hours after flow cytometry analyses, using a magnification of 10x. Footnotes: GFP+, green fluorescent protein positive cells; HRR, homologous recombination repair; WT, wild type; I-SceI, endonuclease causing double strand break; sc siRNA, scrambled siRNA. Supplementary Figure 4. Original western blot images for Supplementary figure 1. The images are not cropped or edited. (A) The figure shows original images from four replicates for the fusion protein DBD-BRCT. Loading order replicate 1: WT, EV, p.Leu1439Phe, p.Phe1668Leu. p.Leu1701Met, p.Ala1708Val, p.Gly1709Arg, p.Lys1711Ser, p.Trp1718Ser, p.Pro1749Ala, not relevant, WT, EV, not relevant*, not relevant, p.Arg1835Gln, not relevant, p.Met1652Ile, p.Val1804Asp, not relevant, p.Arg1699Trp, p.Val1838Gly. Loading order replicate 2: WT, EV, p.Leu1439Phe. p.Met1652Ile, not relevant, p.Leu1701Met, p.Arg1699Trp, p.Leu1701Met, p.Ala1708Val, p.Gly1709Arg, WT, p.Lys1711Ser, not relevant, p.Val1804Asp, not relevant, not relevant, p.Arg1835Gln, not relevant, WT, EV, p.Trp1718Ser, p.Pro1749Ser, p.Val1838Gly. Loading order replicate 3: WT, EV, p.Met1652Ile, p.Val1804Asp, p.Leu1439Phe, p.Phe1668Leu, p.Leu1701Met, p.Ala1708Val, p.Gly1709Arg, p.Lys1711Ser, p.Trp1718Ser, WT, EV, p.Ser1655Phe, p.Arg1699Trp, p.Val1838Gly, p.Pro1749Ala, not relevant, not relevant, not relevant, p.Arg1835Gln, not relevant. Loading order replicate 4: WT, p.Glu1535Lys, p.Met1628Ile, WT, p.Glu1535Lys, p.Met1628Ile, WT, p.Glu1535Lys, p.Met1628Ile. BRCA1 was detected with anti-BRCA1 antibody. The BRCA1 bands were normalised against the total protein of each lane. (B) The figure shows images from four replicates for the full-length His-BRCA1. The blots were cut in two prior to incubation with anti-BRCA1 and anti-actin, so each blot are divided into two images. Loading order replicate 1, 2 and 3: WT, p.Leu1439Phe, p.Glu1535Lys, p.Met1628Ile, p.Phe1668Leu, p.Leu1701Met, p.Ala1708Val, p.Gly1709Arg, p.Lys1711Gn, p.Trp1718Ser, WT, not relevant, not relevant, not relevant, p.Pro1749Ala, p.Arg1835Gln, p.Arg1699Trp, p.Val1838Gly, p.Met1652Ile, p.Val1804Asp. Loading order replicate 4: WT, EV, EV, EV. BRCA1 was detected with anti-BRCA1 antibody. Actin was used as loading control to normalise the corresponding BRCA1 bands. *Samples included in the Western Blot, but not relevant in this study.