Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Influence of BRCA1 on IR-induced phosphorylation and localization of ATM. (A) HCC1937 (BRCA1-deficient) cells were transiently transfected with a human expression vector, carrying (BRCA1wt) or not carrying (vector) a wild-type BRCA1 gene. Following exposure to indicated doses of ionizing irradiation, total and phosphorylated ATM was assessed by immunoblot after immunoprecipitation, and total and phosphorylated SMC1 and total BRCA1 were assessed by immunoblot of whole-cell extracts. (B) Subcellular localization of BRCA1, phophorylated ATM, and H2AXγ in BRCA1-deficient or -proficient cells. Cells were fixed 15 min after 0 Gy (IR-) or 5 Gy (IR+) of γ irradiation, then labeled with antibodies or DAPI as indicated.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Transfection, Expressing, Plasmid Preparation, Irradiation, Western Blot, Immunoprecipitation, Labeling

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Interdependencies of IR-induced foci formation and pbosphorylation of NBS1 and BRCA1. (A,B) Subcellular localization of NBS1 and BRCA1 in HCC1937 (BRCA1-deficient) cells transfected with a transient expression vector carrying BRCA1 gene (BRCA1wt) or an empty vector (vector; A) and NBS1-LB1 (NBS1-deficient) cells stably transfected with a retrovirus vector carrying p95/Nbs1 gene (p95) or an empty vector (vec) were analyzed by immunofluorescence microscopy (B). Cells were fixed 15 min after 0 Gy (IR-) or 5 Gy (IR+) of γ irradiation, then labeled with anti-NBS1 (shown in red) or anti-BRCA1 (shown in green). DNA was visualized by staining with DAPI (shown in blue). (C) HCC1937 (BRCA1-deficient) cells were transiently transfected with a human expression vector, pcDNA3 carrying (BRCA1wt) or not carrying (vector) wild-type BRCA1 gene. Following exposure to indicated doses of ionizing irradiation, cellular extracts were subjected to immunoblotting. Cell extracts were blotted with antibody to phosphorylated Ser 343 of NBS1, then reblotted with anti-NBS1 antibody. (D) Cellular extracts of NBS1-LB1 cells stably transfected with a retrovirus vector carrying p95/NBS1 gene [p95(NBS1)] or an empty vector (vector) exposed to indicated doses of ionizing irradiation were separated on 5% Tris-acetate gel and blotted with anti-BRCA1 antibody.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Transfection, Expressing, Plasmid Preparation, Stable Transfection, Immunofluorescence, Microscopy, Irradiation, Labeling, Staining, Western Blot

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Targeted modification of the mouse Smc1 gene and effects on radiation responses. (A) Schematic of Smc1 knock-in procedure. (panel i) Genomic structure of the mouse Smc1 gene on chromosome X. The whole genomic Smc1 gene consists of 26 exons shown as boxes. Targeted region into which to insert the knock-in vector is shown as a thick bar. (panel ii) Targeting construct of the knock-in vector, pSmc1KI–Neotk, targeted locus, and targeted locus following cre-mediated recombination. Neomycin resistance and thymidine kinase genes (Neo-tk) flanked by loxP sites (shown as black triangles) are inserted into an intron between exon 18 and 19. In pSmc1KI–Neotk, two nucleotide exchanges to change serine to alanine at amino acid positions 957 and 966 are made in exon 19 (shown as a dark box labeled as 19*). Homologus recombination of the pSmc1KI–Neotk is depicted by the large “X”s. Positions of the flanking probe for genotyping and BstEII sites are indicated. (B) Wild-type (Smc1WT) or Smc1S957AS966A immortalized mouse fibroblast cells were exposed to 0 Gy (IR-) or 10 Gy (IR+) of ionizing irradiation and cellular extracts were prepared 30 min later. Whole-cell lysates were subjected to immunoblotting with antibodies as indicated. (C) Thirty minutes after exposure to 0 (-) or 10 (+) Gy of ionizing irradiation (IR), whole-cell lysates were prepared from wild-type or mutant knock-in fibroblasts were immunoblotted with anti-mouse NBS1 or BRCA1 antibodies. Duplicate samples of cell lysates from irradiated knock-in cells were treated with protein phosphatase (PPase+) prior to electrophoresis.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Modification, Knock-In, Plasmid Preparation, Construct, Labeling, Irradiation, Western Blot, Mutagenesis, Electrophoresis

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: SMC1 phosphorylation is not required for IR-induced formation of foci containing phospho-ATM, H2AXγ, NBS1, 53BP1, phosphorylated CHK2, or BRCA1. Wild-type (Smc1WT) or Smc1 phosphorylation mutant knock-in (Smc1S957AS966A) fibroblast cells were fixed with 4% paraformaldehyde 30 min after 0 Gy (-) 10 Gy (+) of ionizing irradiation (IR), then subjected to immunofluorescence microscopy. (A) Staining with antibodies recognizing phosphorylated ATM or SMC1. (B) Staining with antibodies recognizing H2AXγ, NBS1, 53BP1, phosphorylated CHK2, BRCA1, and phosphorylated SMC1. For costaining of phosphorylated SMC1 (red) with BRCA1 (green), rabbit polyclonal anti-Ser957p antibody was used.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Mutagenesis, Knock-In, Irradiation, Immunofluorescence, Microscopy, Staining

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Proposed model for an IR-induced signaling pathway. Chromatin structure changes caused by DNA breakage or other mechanisms leads to intermolecular autophosphorylation of ATM dimers, resulting in release of phosphorylated and active ATM monomers. If DNA-strand breaks are present, several proteins, including NBS1 and BRCA1, are recruited to the sites of the breaks independent of the ATM activation process. After activation, monomeric ATM can phosphorylate nucleoplasmic substrates, like p53, and if NBS1 and BRCA1 have localized to DNA breaks, activated ATM is recruited to the break. At the DNA break, activated ATM can phosphorylate substrates, including SMC1. The phosphorylation of SMC1 reduces chromosomal breakage and enhances cell survival.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Activation Assay

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Figure 6. Histone H2A ubiquitination accompanied by BRCA1 activation is the hallmark phenotype of BRAP LOF (A) Immunoblotting of histone extracts from NPCs as well as from embryonic, neonatal, adult cerebral cortical tissues, and quantification (Mean G SD) of increases in histone H2Aub (total H2Aub and H2AubK119, respectively) resulted from Brap LOF. n = 3–6 biological replicates. p-values calculated by Student’s t test are indicated. (B) Immunoblotting of Brca1 in various cells and tissues, showing that Brap LOF results in increased Brca1 abundance. (C) Immunoblotting of nuclear vs cytoplasmic fractions of MEFs at P1, showing increased nuclear localization of Brca1 in Brap/ cells. (D) Brca1 (red) and NeuN (green) double immunohistology images of cerebral cortical sections of BrapcKONPC and control mice at four months of age. Representative images are shown. Note the increased intensity and density of Brca1 puncta in the nuclei of BrapcKONPC cortical neurons (NeuN+). (E and F) Immunoblotting analyses of histone extracts from cerebral cortical tissues of three-month-old mice, showing increased ubiquitination of H2A variants targeted by Brca1 (E) along with total histone H2A ubiquitination (F). (G) Double immunohistology staining of cortical sections of 4-month old WT or BrapcKONPC mice with antibodies against Gfap (green) and histone H3 (red), showing reduced nuclear histones in cells surrounded by reactive astrocytes (circles) in BrapcKONPC cortical tissues. Representative images are shown. Nuclear DNA was stained with Hoechst 33342. Bars: 50 um or as indicated. See also Figure S3.

Article Snippet: Phospho-p53 (Ser15) Abcam Cat# ab1431; RRID:AB_301090 Phospho-p53 (Ser15) (D4S1H) Cell Signaling Technology Cat# 12571; RRID:AB_2714036 Phospho-p53 (Ser15) (16G8) Cell Signaling Technology Cat# 9286; RRID:AB_331741 Phospho-ATM(Ser1981) Novus Biologicals Cat# AF1655 Phospho-ATR (Ser 428) Cell Signaling Technology Cat# 2853; RRID:AB_2290281 53 BP1 Novus Biologicals Cat# NB100-304 Brca1 Santa Cruz Biotechnology Cat# sc-642; RRID:AB_630944 Brca1 Novus Biologicals Cat# MAB22101 Brca1 (clone 8F7) Novus Biologicals Cat# NBP1-41186 LC3B (G-9) Santa Cruz Biotechnology Cat# sc-376404; RRID:AB_11150489 LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 LC3B Novus Biologicals Cat# NB100-2220 p62SQSTM1 Novus Biologicals Cat# MAB8028 p62SQSTM1 GeneTex Cat# GTX100685; RRID:AB_2038029

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Staining

Journal: Scientific Reports

Article Title: TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

doi: 10.1038/srep04663

Figure Lengend Snippet: (a) Immunofluorescence showing the ΔNp63 protein (top row) in MCF7 cells treated as indicated above. BRCA1 protein (second row) is readily detectable in control cells, but disappears after treatments promoting expression of ΔNp63. MDM2 protein (third row) correlates with the expression of BRCA1. (b) qRT-PCR showing fold change in mRNA levels for ΔNp63, MDM2, and BRCA1 following treatments indicated above. (c) Western blot confirming an effective reduction of BRCA1 protein level and stabilization of the TP53 protein after treatments indicated above. F12, cells were cultured in DMEM/F12 medium instead of the regular DMEM; Serdemetan, cells treated with MDM2 inhibitor as described in Materials and Methods; siBRCA1 and siMDM2, cells treated with siRNAs targeting corresponding genes. Scale bars correspond to 20 µm.

Article Snippet: Primary antibodies were used as follows: goat anti-TAp63 (Santa Cruz, sc-8608, diluted 1:50), rabbit anti-Ki67 (Abcam, ab15580, diluted 1:200), goat anti-ΔNp63 (Santa Cruz, sc-8609, diluted 1:100), rabbit anti-MDM2 (Abcam, ab58530, diluted 1:100), mouse monoclonal anti-Krt18 (Abcam, ab668, diluted 1:100), mouse monoclonal anti-p53 (Abcam, ab26, diluted 1:100), mouse monoclonal anti-p53 (Abcam, PAb 240, diluted 1:100), mouse anti-human BRCA1 (Calbiochem, OP92, diluted 1:100), and mouse monoclonal anti-Nucleophosmin (Invitrogen, FC-61991, diluted 1:200, a kind gift of Karita Peltonen and Prof. Marikki Laiho).

Techniques: Immunofluorescence, Control, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

doi: 10.1038/srep04663

Figure Lengend Snippet: (a) Untreated MCF10A cells express high levels of ΔNp63, and low levels of BRCA1 and MDM2. Cells plated at a low density translocate ΔNp63 to nucleoli, and elevate expression of BRCA1, and MDM2. (b) Knockdown of TP63 (siTP63) results in upregulation of BRCA1 and MDM2. (c) Western blot confirming an efficient siRNA-mediated knockdown of BRCA1 and ΔNp63 proteins, and lack of TP53 in TP53 −/− MCF10A cells. Notice a higher level of ΔNp63 protein in BRCA1-depleted TP53-mutant cells. Asterisk indicates a 75 kDa band from a molecular weight marker loaded in the same lane as the siMDM2 sample. (d) TP53-deficiency in MCF10A cells leads to an increase in MDM2 mRNA level as measured by qRT-PCR. ( e ) Immunofluorescence staining of TP53 −/− and its parental isogenic TP53 +/+ MCF10A cell lines demonstrates a ubiquitous relocation of ΔNp63 protein from the nucleoplasm in TP53 +/+ cells (arrowheads) to nucleoli in TP53 −/− cells (arrows), and upregulation of BRCA1 in TP53 −/− cells. Insets illustrate the differences at a higher magnification. Scale bars correspond to 20 µm.

Article Snippet: Primary antibodies were used as follows: goat anti-TAp63 (Santa Cruz, sc-8608, diluted 1:50), rabbit anti-Ki67 (Abcam, ab15580, diluted 1:200), goat anti-ΔNp63 (Santa Cruz, sc-8609, diluted 1:100), rabbit anti-MDM2 (Abcam, ab58530, diluted 1:100), mouse monoclonal anti-Krt18 (Abcam, ab668, diluted 1:100), mouse monoclonal anti-p53 (Abcam, ab26, diluted 1:100), mouse monoclonal anti-p53 (Abcam, PAb 240, diluted 1:100), mouse anti-human BRCA1 (Calbiochem, OP92, diluted 1:100), and mouse monoclonal anti-Nucleophosmin (Invitrogen, FC-61991, diluted 1:200, a kind gift of Karita Peltonen and Prof. Marikki Laiho).

Techniques: Expressing, Knockdown, Western Blot, Mutagenesis, Molecular Weight, Marker, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

doi: 10.1038/srep04663

Figure Lengend Snippet: TP53 prevents translocation of ΔNp63 from the nucleoplasm into nucleoli. Active TP53 and a nucleoplasmic ΔNp63 inversely correlate with MDM2, BRCA1, and TAp63. MDM2 and BRCA1 in luminal cells inhibit TP53, thus preventing the nucleoplasmic expression of ΔNp63 and suppressing the basal-like differentiation. Loss of TP53 leads to an upregulation of TAp63, which is associated with an unlimited cell proliferation rather than a particular epithelial subtype. Relocation of ΔNp63 to nucleoli in basal epithelial cells is associated with EMT.

Article Snippet: Primary antibodies were used as follows: goat anti-TAp63 (Santa Cruz, sc-8608, diluted 1:50), rabbit anti-Ki67 (Abcam, ab15580, diluted 1:200), goat anti-ΔNp63 (Santa Cruz, sc-8609, diluted 1:100), rabbit anti-MDM2 (Abcam, ab58530, diluted 1:100), mouse monoclonal anti-Krt18 (Abcam, ab668, diluted 1:100), mouse monoclonal anti-p53 (Abcam, ab26, diluted 1:100), mouse monoclonal anti-p53 (Abcam, PAb 240, diluted 1:100), mouse anti-human BRCA1 (Calbiochem, OP92, diluted 1:100), and mouse monoclonal anti-Nucleophosmin (Invitrogen, FC-61991, diluted 1:200, a kind gift of Karita Peltonen and Prof. Marikki Laiho).

Techniques: Translocation Assay, Expressing