Journal: Molecular Medicine Reports

Article Title: LncRNA AWPPH promotes the proliferation, migration and invasion of ovarian carcinoma cells via activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/mmr.2019.10029

Figure Lengend Snippet: Effects of AWPPH overexpression on β-catenin expression. Reverse transcription-quantitative polymerase chain reaction and western blot analyses revealed elevated levels of (A) AWPPH mRNA and (B) β-catenin protein expression in UWB1.289 and UWB1.289 + BRCA1 cells following transfection with AWPPH plasmid, compared with cells transfected with NC vector and non-transfected cells. Western blotting was performed in triplicate. Data are presented as the mean ± standard deviation. *P<0.05. AWPPH, associated with poor prognosis of hepatocellular carcinoma; NC, negative control vector; OC, ovarian carcinoma.

Article Snippet: A total of two human OC cell lines, UWB1.289 (CRL-2945TM) and UWB1.289 + BRCA1 (CRL-2946TM), were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Standard Deviation, Negative Control

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: Cancer research

Article Title: Phosphatase 1 nuclear targeting subunit mediates recruitment and function of poly (ADP-ribose) polymerase 1 in DNA repair

doi: 10.1158/0008-5472.CAN-18-1673

Figure Lengend Snippet: (A) HeLa cells were transfected with control, non-targeting, or PNUTS siRNA (#1) for 1 day. The cells were then treated with IR at the indicated doses, incubated for 30 min, harvested and analyzed by immunoblotting for γH2AX, PNUTS, and H2B. (B) HeLa cells were transfected with PNUTS siRNA (#1), and siRNA-resistant (SiR) WT PNUTS, as indicated. The cell lysates were analyzed by immunoblotting for γH2AX, PNUTS, and β-actin. (C, D) HeLa cells were treated with PNUTS siRNA (#1 and #2), as indicated. The comet assay was performed as described in Materials and Methods. Representative images are shown in panel C. The percentage of DNA in the tail section was quantified, the mean values and standard derivations are shown in panel D (N>20). (E) HeLa cells were treated with control or PNUTS siRNA (#1) for 1 day. The cell lysates were analyzed by immunoblotting for γH2AX, phospho-CHK1, phospho-BRCA1, PNUTS, and β-actin. (F) SCC38 cells were transfected with PNUTS siRNA (#1), and siRNA-resistant (SiR) WT or W401A PNUTS, as indicated. The cell lysates were analyzed by immunoblotting for γH2AX, PNUTS, and β-actin. (G) SCC38 cells were treated with control or PNUTS siRNA (#1) at day 0, incubated with doxorubicin (Dox, 2 μg/ml) at day 1, and incubated for 3 days. Cell viability was determined and normalized to that of day 1. The mean value and standard deviation were calculated from 3 independent experiments. (H) SCC38 cells were treated with PNUTS siRNA (#1) and siRNA-resistant (SiR) PNUTS, as in panel G. These cells were then treated with Dox (2 μg/ml), and incubated for 1-4 days. Cell viability was determined and normalized to that of the first day. The mean value and standard deviation were calculated from 3 independent experiments. (I) The colonogenic assay was performed as described in Materials and Methods. The numbers of colonies formed were normalized to the untreated control. The mean value and standard deviation were calculated from 3 independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value<0.001 was considered highly significant (***).

Article Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as previously described ( 25 ), using the following antibodies:, γH2AX, PNUTS, PARP1, H2B, BRCA1 phospho-S1524, CHK1 phospho-S317 antibodies from Bethyl Laboratories; β-actin, GFP, Poly (ADP-Ribose) Polymer antibodies from Abcam; α-tubulin, BRCA1 phospho-S1524, active caspase-3 antibodies from Cell Signaling Technology.

Techniques: Transfection, Incubation, Western Blot, Single Cell Gel Electrophoresis, Standard Deviation

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