Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: The level of the interaction between cBRC3 and cRAD51 was half of that observed with cBRC4 and similar to that with hBRC3 and hBRC4. The interaction between hRAD51 and cBRC3 was stronger than with hBRC3. (A) VP16 transactivation domain-fused cRAD51 construct or empty vector and a Gal4-DBD-fused cBRC3, cBRC4, hBRC3, hBRC4, or cBRCA2 N-terminus (1–1000 aa) were introduced into HeLa cells to determine their interaction with cRAD51 in a mammalian two-hybrid luciferase-based assay. HeLa cells were co-transfected with the BRC repeat constructs and cRAD51 expression vector constructs with the reporter plasmids pGluc and pRL-TK. The pRL-TK construct was used to normalize the transfection efficiency. Lysate luciferase activity was determined 48 h after transfection. (B) same as (A), except that VP16 transactivation domain-fused hRAD51 was used. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test, followed by a Tukey's post-test. Different letters indicate significant differences between the cells transfected with the indicated constructs. (p<0.01). DBD, DNA-binding domain; TAD, transactivation domain-fused proteins.

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Construct, Plasmid Preparation, Luciferase, Transfection, Expressing, Activity Assay, Binding Assay

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) Amino acid sequence alignment of BRC3 among canines (Accession No. NP_001006654.2), humans (Accession No. NP_000050.2), and mice (Accession No. NP_033895.2). (B) Amino acid sequence alignment of cBRC3 and hBRC4. The pink characters indicate the positions of missense mutations in cBRC3. (C–E) The contacts between the residues of 1526T (C), T1526P (D), or T1526A (E) and hRAD51 or other residues of BRC4 were calculated and are depicted. Human BRC4 and hRAD51 are depicted by gold and pink ribbons, respectively. Solid green lines signify stable contacts as determined by the University of California, San Francisco Chimera software.

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Sequencing, Software

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) The GST pull-down assay showed that wild-type and K1435R mutant canine BRC3 (cBRC3) interacted with RAD51, but the T1425P mutant cBRC3 did not interact with RAD51. GST-fused peptides were incubated with human RAD51 (hRAD51) at 4°C for 1 h. Equilibrated beads were incubated with the protein–peptide complexes at 4°C for 1 h. Complexes bound to the beads were thoroughly washed. The samples were analyzed by SDS-PAGE and western blotting with anti-RAD51 IgG, or anti-GST IgG, and horseradish peroxidase conjugated anti-rabbit IgG. Blots were developed using the ECL Plus Western Blotting Detection System. (B) Schematic of the enzyme-linked immunosorbent assay (ELISA) used to detect the cBRC-hRAD51 interaction and its disruption by soluble peptides. (C) In a competitive ELISA, GST-cBRC3 with the T1425P mutation did not inhibit the solid-phase interaction between GST-cBRC3 and hRAD51. However, the substitution of GST-cBRC3 with the K1435R mutation showed no interaction with hRAD51, like wild-type cBRC3. Values are expressed as the mean percentage absorbance at 405 nm for data sets in triplicate normalized to the positive control, which was not treated with the soluble inhibitors, and the negative control, which was coated with GST and treated with hRAD51. Significance was examined by a one-way analysis of variance test followed by Tukey's post-test. The asterisks indicate a significant difference compared with GST-treated data sets at the same competitor concentration (p<0.01).

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Pull Down Assay, Mutagenesis, Incubation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Positive Control, Negative Control, Concentration Assay

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: The binding intensity of T1425P mutant canine BRC3 (cBRC3) and canine RAD51 (cRAD51) or human RAD51 (hRAD51) was much lower than that of wild-type cBRC3 and cRAD51 or hRAD51, while that of the K1435R mutant cBRC3 and cRAD51 or hRAD51 was higher than between wild-type cBRC3 and cRAD51 or hRAD51. (A) Gal4-DNA binding domain-fused wild-type and mutant cBRC3 or mutants of human BRC4 (hBRC4) and VP16 transactivation domain-fused cRAD51 constructs were introduced into HeLa cells to determine their interaction with cRAD51 in a mammalian two-hybrid assay measuring luciferase activity. HeLa cells were co-transfected with the BRC repeat constructs and cRAD51 expression vector constructs and with the reporter plasmids pGluc and pRL-TK. The pRL-TK construct was used to normalize the transfection efficiency. Lysate luciferase activity was determined 48 h after transfection. (B) same as (A), except that VP16 transactivation domain-fused hRAD51 was used. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared with cells transfected with wild-type (WT) cBRC3 (p<0.01).

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Binding Assay, Mutagenesis, Construct, Two Hybrid Assay, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) Schematic of the modified mammalian two-hybrid assay used to examine how mutant canine BRC3 (cBRC3) interferes with the RAD51-RAD51 interaction. (B) The graph shows the strength of the interference caused by the expression of EGFP empty vector, wild-type EGFP-cBRC3, 2 mutants of cBRC3 (T1425P and K1435R), and cBRCA2 N-terminus (1–1000 aa) on the canine RAD51 (cRAD51)-cRAD51 interaction as determined by the modified mammalian two-hybrid assay. Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared with the cells transfected with an empty vector (*: p<0.05, **: p<0.01). The negative control for this assay is shown in Supporting information . (C) same as (B), except that human RAD51 (hRAD51) was used. (D) Immunostained cells, which were transiently transfected with cBRC3 mutants, after being subjected to ionizing radiation, are shown. The indicated cells were irradiated (15 Gy) and fixed 12 h after exposure to the ionizing radiation. (E) HeLa cells transfected with wild-type or mutant cBRC3 or empty vector were irradiated (15 Gy) and then allowed to recover for the times indicated. Images containing at least 100 cells were captured by a computer, and the number of cells containing at least 10 foci were recorded and plotted as a percentage of the total number of cells. The plots were generated from 3 independent experiments. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared with irradiated cells transfected with an empty vector at the same time point (p<0.01).

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Modification, Two Hybrid Assay, Mutagenesis, Expressing, Plasmid Preparation, Transfection, Negative Control, Irradiation, Generated

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) Amino acid sequence alignment among canine BRC3 (cBRC3) (Accession No. NP_001006654.2), wild-type human BRC3 (hBRC3) (Accession No. NP_000050.2), and 2 mutants of hBRC3. The pink characters indicate the positions of missense mutations in cBRC3. (B) Gal4-DNA binding domain-fused wild-type and mutant hBRC3 and VP16 transactivation domain-fused cRAD51 constructs were introduced into HeLa cells to determine their interaction with cRAD51 in a mammalian two-hybrid assay measuring luciferase activity. HeLa cells were co-transfected with the hBRC3 constructs and canine RAD51 (cRAD51) expression vector constructs and with the reporter plasmids pGluc and pRL-TK. The pRL-TK construct was used to normalize the transfection efficiency. Lysate luciferase activity was determined 48 h after transfection. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared to cells transfected with wild-type (WT) cBRC3 (*: p<0.05, **: p<0.01). (C) same as (B), except that VP16 transactivation domain-fused hRAD51 was used. (D) The graph shows the strength of the interference caused by the expression of EGFP empty vector, wild-type EGFP-hBRC3, 3 mutants of hBRC3 (K1440R, K1440E, and T1430P), and cBRCA2 N-terminus (1–1000 aa) on the cRAD51-cRAD51 interaction as determined by the modified mammalian two-hybrid assay. Significance was examined by one-way ANOVA test followed by Dunnett's test. The asterisks indicate a significant difference compared to the cells transfected with an empty vector (*: p<0.05, **: p<0.01). The negative control for this assay is shown in Supporting information . (E) same as (D), except that hRAD51 was used.

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Sequencing, Binding Assay, Mutagenesis, Construct, Two Hybrid Assay, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation, Modification, Negative Control

Journal: Cancers

Article Title: The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis

doi: 10.3390/cancers13225746

Figure Lengend Snippet: Impact of deletion of MUC4 EGF1 and MUC4 EGF2 domains on binding affinities with HER2 and on proliferation and migration properties of human PC cells. ( A ) Schematic representation of the membrane-bound mucin MUC4 featuring both subunits MUC4α and MUC4β. MUC4α is rich in proline/serine/threonine residues (PST domain), is strongly O -glycosylated, and contains the AMOP and the NIDO domains. The subunits are post-transcriptionally cleaved by an auto-cleavable GDPH sequence and noncovalently associated. MUC4β is formed by a von Willebrand factor-D (VWD), an uncharacterized strongly N -glycosylated part, a cysteine-rich domain (CR), two EGF-like domains (EGF3 and EGF1), an intermediate uncharacterized domain (DI), another EGF-like domain (EGF2), and a transmembrane helix (TM) with a short cytoplasmic tail (CT). ( B ) Measurement of binding affinities between HER2 and eGFP-MUC4β ΔTM containing the EGF domains (MUC4β ΔTM ) and the deleted forms for each EGF domain: eGFP-MUC4β ΔEGF1 (MUC4 ΔEGF1 ), eGFP-MUC4β ΔEGF2 (MUC4 ΔEGF2 ), or eGFP alone (mock, negative control), using MST. Measurement of Panc-1 ( C ) and MiaPaCa-2 ( D ) cell proliferation after transfection with eGFP-MUC4β containing the EGF domains and the deleted forms for each domain: MUC4β ΔEGF1 , MUC4β ΔEGF2 , or eGFP alone (negative control). Proliferation ratio over eGFP was calculated for each construct. Measurement of cell migration with Boyden chamber (control insert) ( E ) and cell invasion with Boyden chamber (Matrigel ® insert) ( F ) in Panc-1 and MiaPaCa-2 cells after transfection with eGFP-MUC4β, the deleted forms MUC4β ΔEGF1 and MUC4β ΔEGF2 , or eGFP alone (negative control). Statistically significant differences between eGFP-MUC4β and the deleted forms of MUC4 or eGFP alone are indicated; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: In order to obtain a monomeric eGFP-MUC4β fusion protein (from #I1447-#P2169, optimized for CHO-K1 expression), only the β subunit was kept, except for the four aa involved in the cleavage (GDPH) (ProteoGenix SAS, Schiltigheim, France).

Techniques: Binding Assay, Migration, Sequencing, Negative Control, Transfection, Construct