Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Eight out of ten constructs successfully modified for expression-pattern characterization in mice

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Construct, Modification, Expressing

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Eight novel Hprt targeted embryonic stem cell lines successfully generated

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Clone Assay, Isolation

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Summary of expression pattern from reporter mouse strains

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Expressing

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Human bacterial artificial chromosomes can be targeted at Hprt by homologous recombination and, if desired, conditionally removed using cre recombinase. (a) Integration into the mouse genome of the bacterial artificial chromosome (BAC)- lacZ -reporter constructs by homologous recombination results in the human gene in either direction relative to the X chromosome; this schematic presents one possible orientation. Regardless of orientation, each insertion resulted in the presence of four loxP sites in the genome (two wild-type and one 511 mutant at one end and one wild-type at the other end of the BAC insert). (b) Crossing the BAC- lacZ- reporter females to ACTB-cre males should result in the generation of two different male offspring; BAC- lacZ -reporter animals, wild-type for the ACTB-cre transgene; and BAC- lacZ- reporter animals carrying the ACTB-cre transgene. Only the reporter animals that are positive for the ACTB-cre gene should recombine the outer most loxP sites, resulting in excision of the BAC construct from the genome and leaving one loxP site. This would result in an absence of lacZ- positive signal. hP , human HPRT promoter; h1, human first exon; m2 and m3, mouse second and third exons; mouse homology arms (dark blue); Hprt coding regions (red); vector backbone (yellow with black edges); Sac B gene from BAC vector backbone (brown); 5′ and 3′ untranslated regions of the human gene (orange); coding region of the human gene (green); lacZ reporter gene (light blue). Schematic, not to scale. (c-f) lacZ expression results from AMOTL1 - lacZ , MAOA - lacZ , NOV - lacZ , and NR2F2 - lacZ females bred to the ACTB-cre males are presented. lacZ -positive staining (blue) was detected in AMOTL1 - lacZ , MAOA - lacZ , NOV - lacZ , and NR2F2 - lacZ males not carrying the ACTB-cre allele whereas absence of staining was detected in males positive for ACTB-cre by genotyping ( AMOTL1 - lacZ , ACTB-cre; MAOA - lacZ , ACTB-cre; NOV - lacZ , ACTB-cre; NR2F2 - lacZ , ACTB-cre), suggesting whole BAC excision from the genome. Scale bar: (c-f) 1 mm. N = 3 animals for all genotypes.

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Homologous Recombination, Construct, Mutagenesis, Plasmid Preparation, Expressing, Staining

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Human NR2F2 - lacZ expressed in mature neurons populating the basolateral and corticolateral amygdaloid nuclei that are immunoreactive for the Nr2f2 mouse protein. Expression analysis of the human NR2F2 - lacZ strain was undertaken by examination of β-galactosidase (β-gal) staining (blue). (a) E12.5 whole embryos revealed staining in the rostral secondary prosencephalon (black arrow) that extended throughout all three prosomeric regions of the diencephalon (black arrowhead). Staining was present in the nasal cavity (white arrow), the vestibulochochlear ganglion (red arrowhead) and mesenchyme of the posterior limbs (white arrowhead). (b) E12.5 cleared embryos additionally demonstrated staining in the developing bladder (black arrow). (c) P7 brains stained in the amygdala nuclei (white arrows), and the subthalamic nuclei (black arrows). (d) Adult brains revealed strong staining extending from the posterior basolateral amygdaloid nuclei (BLP) (black arrows) to the posterolateral cortical amygdaloid nuclei (PLCo) (white arrows), and the posteroventral part of the medial amygdaloid nuclei (MePV) (white arrowheads). Broad staining was detected in the ventral thalamic area, excluding the cerebral peduncle (cp). (e) Colocalization experiment using β-gal staining and a neuronal nuclei (NeuN) antibody (brown) performed on adult brain cryosections revealed strong expression of NR2F2 - lacZ in mature neurons populating the BLP, and the basomedial amygdaloid nuclei (BMP) (red arrows). Colocalization was found in the PLCo, and the posteromedial cortical amygdaloid nuclei (PMCo) (black arrows), and the MePV (black arrowhead). Lower level of β-gal staining was detected in mature neurons in the anterolateral amygdalohippocampal area (AHiAL) (red arrowhead). Boxed region in (e) is shown in (f) . (f) Higher magnification revealed strong expression of β-gal in mature neurons in the PMCo and sparse expression in mature neurons in the AHiAL. (g) Colocalization experiment, using an anti-β-gal antibody (green), and an NR2F2 antibody (red), performed on adult brain cryosections revealed strong β-gal labeling in cells expressing the Nr2f2 mouse gene in brain regions extending from the PMCo (white arrowhead) to the MePV (white arrow). Lower levels of β-gal were detected in the AHiAL (red arrow). Boxed region in (g) is shown in (h) . (h) Higher magnification revealed strong expression of β-gal (green) in Nr2f2 -positive cells (red) in the PMCo (white arrow) and lower expression in the AHiAL (white arrowhead). LV, lateral ventricle. Scale bar: (a - d) 1 mm; (e , g) 100 μm; (f , h) 20 μm.

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Expressing, Staining, Labeling

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Comparative genomics delineated the DNA boundaries that were sufficient for adult brain-specific expression of NOV and NR2F2 . Coordinates corresponding to the human bacterial artificial chromosome (BAC) constructs used in this study were retrieved and visualized using the University of California Santa Cruz (UCSC) genome browser. (a) DNA alignment of the human NOV BAC (RP11-840I14) (black), against both the RP23-235B13 BAC construct used in the Gene Expression Nervous System Atlas (GENSAT) mouse model (blue), and the mouse genome, delineated the genomic DNA boundaries sufficient for proper expression of this human gene in the basolateral amygdaloid nuclei, cortical layers, and pyramidal neurons in the cornu ammonis 1 (CA1) regions in the adult brain. One hypothesis suggested by our results was that additional functionally conserved regulatory elements homologous to the large non-overlapping 3′ mouse-BAC region are necessary for proper human-gene expression in the developing cortical layers at P7. (b) DNA alignment of the human NR2F2 BAC (RP11-134D15) (black), against both the RP23-109L9 BAC construct used in the GENSAT mouse model (blue) and the mouse genome, delineated the genomic DNA boundaries sufficient for region-specific expression of this human gene in the basolateral, and corticolateral amygdaloid nuclei in the adult brain. One hypothesis suggested by our results was that additional functionally-conserved regulatory elements homologous to the non-overlapping 5′ mouse-BAC region are necessary for proper expression in the developing hypothalamus at P7. Black rectangle box in (b) is shown in (c) . (c) Sequence alignment using the coordinates of the primers used in the BAC lacZ retrofitting process (grey bars) and the cDNA sequence used to generate an anti-NR2F2 antibody (black bar), suggested that the absence of expression of the NR2F2 - lacZ constructs in retinal amacrine cells was not attributable to detection of different isoforms of NR2F2 .

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Expressing, Construct, Gene Expression, Sequencing

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Primers used for reporter-gene retrofitting

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Sequencing

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Structure–function analysis of BCL11A defines a region of BCL11A that interacts with NTHL1 and stimulates its enzymatic activity. ( A ) Diagrammatic representation of the BCL11A-XL and fragments tested in pull-down and DNA repair assays. ( B ) Diagrammatic representation of the 5,6-dihydrothymidine (DHT) cleavage assay using a fluorophore reporter probe. The assay was performed using 5 nM of NTHL1 and 20 nM of BCL11A-XL, in the presence of 50 nM of BSA. ( C ) Diagrammatic representation of the thymine glycol (Tg) cleavage assay using a radioactively labeled probe. The assay was performed using purified NTHL1 (10 nM) and the indicated amounts of BSA and/or BCL11A-XL. The reaction in lane 1 was performed with a probe that contains a normal thymidine base instead of thymine glycol. ( D ) Pull-down assays were performed using His-tagged BCL11A fragments and either GST or GST-NTHL1, followed by immunoblotting with anti-His antibodies. ( E ) DHT cleavage assays were performed using 20 nM of BCL11A fragments, in the presence of absence of NTHL1 (5 nM), as indicated. An additional assay was performed with 40 nM of BCL11A 160–520 . All samples included 50 nM of BSA; the sample labeled ‘BSA’ included an additional 20 nM BSA. ( F ) Thymine-glycol (Tg) cleavage assays were performed using radioactively end-labeled double-stranded oligonucleotides containing a Tg oxidized base, and 250 nM BSA. Where indicated, NTHL1 (2.5 nM), BSA or BCL11A 160–520 (15 nM) were included in the reaction. Production of a cleaved product in samples that have not been treated with NaOH (lanes 1–4) requires the glycosylase and AP/lyase activities of NTHL1. In the absence of NaOH, NTHL1 alone cleaves 9.7% of the substrate, whereas BCL11A 160–520 cleaves 49%. In the presence of NaOH, NTHL1 alone cleaves 20.3% of the substrate, whereas BCL11A 160–520 cleaves 43.8%. ( G ) Radioactively end-labeled double-stranded oligonucleotides containing a thymine glycol (Tg) base were incubated with the indicated amount of NTHL1, BSA and BCL11A 160–520 . After incubation at 37°C, 50 mM sodium borohydride was added. The reactions were incubated for another 15 min at 37°C. After termination of the reaction, the trapped complexes were separated from free DNA by 10% SDS-PAGE. ( H ) Pull-down assays were performed using the His-tagged BCL11A 161-366 peptide and either GST or GST-NTHL1 1-312 , GST-NTHL1 88-312 or GST-NTHL1 1-116 followed by immunoblotting with anti-His antibodies.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Activity Assay, Cleavage Assay, Labeling, Purification, Western Blot, Incubation, SDS Page

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: The DNA repair and proliferation defects caused by BCL11A knockdown are rescued by ectopic expression of the BCL11A 160–520 Fragment. ( A , B , E and F ) MDA-MB-231 cells were infected with lentiviral vectors as indicated: an empty vector, a vector expressing BCL11A shRNA, and a vector expressing BCL11A 160–520 . ( A ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( B ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH 10 after treatment of cells with the Endo III DNA glycosylase. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( C ) MDA-MB-231 cells stably carrying an empty vector or a vector expressing BCL11A 160–520 were transfected with either of two distinct BCL11A dicer RNAs or a control dicer RNA. Genomic DNA was purified and abasic sites were quantified using an aldehyde-reactive probe. Results are an average of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( D ) MDA-MB-231 cells were stably infected with a vector expressing BCL11A shRNA under the control of a tetracycline-inducible promoter, as well as an empty vector or a vector expressing BCL11A 160–520 . Cells were treated or not with doxycycline for 3 days before cell extracts were prepared and analyzed in a DNA repair assay using as a substrate double-stranded oligonucleotide containing a thymine glycol base (Tg). Upon incubation in the presence of 32 P-dTTP, NTHL1 present in the cell extract removes the thymine glycol and introduces a single-strand break; Pol β adds a radioactively labeled thymidine and ligase III seals the strand-break thereby generating a single-strand that migrates more slowly on a denaturing gel. The amount of repair completion (top band) for each sample was normalized to the value of ‘Vector no dox’. The enzymatic activity of the BCL11A knockdown (‘vector + dox’) is 55.7% whereas the activity of ‘BCL11A 160–520 no dox’ is 365% and ‘BCL11A 160–520 + dox’ is 361%. ( E ) Cells were treated with increasing amounts of H 2 O 2 and then submitted to a clonogenic assay. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( F ) MDA-MB-231 cells carrying BCL11A 160–520 or not were infected with lentiviruses expressing either control or BCL11A shRNA. β-Gal associated senescence was measured 5 days after infection. All values are normalized to the control shRNA value. Error bars represent standard error. Results are a representative of one of two different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( G ) MDA-MB-231 cells were stably infected with a lentiviral vector expressing a BCL11A shRNA under the control of a doxycycline-inducible promoter and either an empty vector or a vector expressing BCL11A 160–520 . Doxycycline was added to the medium or not and 3 days later cell proliferation was measured by staining with CellTrace™ CFSE. CFSE was added to the medium and a portion of the population was fixed immediately as the ‘0’ generation. The remaining cells were allowed to proliferate for 3 days. Cells were fixed and analyzed by flow cytometry. Small peaks within the CFSE profiles represent successive generations, as indicated above the peaks. The proliferation index is the total number of divisions divided by the number of cells that went into division. The division index is the average number of cell divisions, taking into account the cells that never divided.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Single Cell Gel Electrophoresis, Stable Transfection, Transfection, Purification, Incubation, Labeling, Activity Assay, Clonogenic Assay, Staining, Flow Cytometry

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A interacts with Pol β and stimulates its enzymatic activities. ( A ) Nuclear protein extracts (500 μg) from MDA-MB-231 cells were submitted to co-immunoprecipitation (IP) alternatively with Pol β or BCL11A antibodies and then immunoblotted (IB) with either anti-Polβ or anti-BCL11A antibody. Input (1%) was loaded as a protein expression control. ( B ) 293 cells were transfected with vectors expressing fusion proteins containing either the N-terminal or C-terminal portion of Intein, as indicated: BCL11A 160–520 -V5-IN4b with either IC2-Flag-LIG3, IC2-Flag-FOXN2 or IC2-Flag-Pol β. Whole cell extracts were submitted to immunoblotting analysis with the V5 and FLAG antibodies. ( C ) Pull-down assays were performed using His-tagged BCL11A fragments and either GST or GST-Pol β, followed by immunoblotting with anti-His antibodies. ( D ) Diagrammatic representation of the strand displacement assay using a fluorophore reporter probe. F: FAM fluorophore; Q: quencher. The strand displacement assay was performed using 2.5 nM of BCL11A and 15 nM of Pol β. 50 nM of BSA were added to each reaction. ( E ) Double-stranded oligonucleotides containing a uracil residue were incubated with UDG and APE1 to form a gapped substrate for Pol β. The DNA was then incubated with 32 P-dCTP in the presence of DNA Pol β and either BSA or BCL11A 160–520 . ( F ) Double-stranded oligonucleotides containing a uracil residue were labeled at the 5'-end and incubated with UDG and APE1 to form a gapped substrate for Pol β. The gapped probe was then incubated with all 4 dNTPs in the presence of DNA Pol β and either 25 nM BSA or 25 nM BCL11A 160–520 . ( G ) Double-stranded oligonucleotides containing a uracil residue, that were labeled at the 3′ end using Klenow and fluorescent CF 660R dCTP, were incubated with UDG and APE1 to produce a single-strand nick with a 5′-deoxyribose phosphate (dRP). This DNA substrate was incubated with Pol β in the presence of BSA or BCL11A 160–520 . NaBH4 was added to most samples, except in lane 9, to prevent the spontaneous conversion of dRP into P. From four independent pairs of assays, we calculated an average 1.8 fold (±0.3) stimulation of Pol β dRP-lyase activity by BCL11A 160–520 .

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot, Incubation, Labeling, Activity Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: The impact of BCL11A knockdown on DNA repair, genomic DNA damage and clonogenic efficiency is confirmed in other triple-negative breast cancer cells. (A and B) The triple-negative breast cancer cell lines BT549, MDA-MB-468 and Hs578T were transfected with either of two distinct BCL11A dicer RNAs or a control dicer RNA. After 2 days, cell extracts were prepared and in parallel cells were submitted to a comet assay. ( A ) Cell extracts were prepared and analyzed in a DNA repair assay with a probe containing a thymine glycol, as described in Figure . ( B ) A fraction of the cells was submitted to single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) Cells were stably infected with a lentiviral vector expressing a BCL11A shRNA under the control of a doxycycline-inducible promoter. Doxycycline was added to the medium or not and 3 days later cells were treated with the indicated concentrations (0, 20, 50) of H 2 O 2 and then submitted to a clonogenic assay. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Transfection, Single Cell Gel Electrophoresis, Stable Transfection, Infection, Plasmid Preparation, Expressing, shRNA, Clonogenic Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Proximity Biotinylation with NTHL1-BirA*. ( A ) Flow chart of manipulations for the BioID experiment. Following treatment with tetracycline and addition of biotin to the medium, cells were treated (2 Gy) or not (0 Gy) with ionizing radiation prior to cell lysis, affinity purification on streptavidin beads and mass spectrometry identification. ( B ) Immunoblotting analysis of Flp-In™ T-Rex™ 293 cells that were engineered to stably carry vectors that express BirA*-FLAG or NTHL1-BirA*-FLAG upon tetracycline induction. ( C ) Immunoblotting analysis with streptavidin confirming the expression of BirA* fusion proteins and successful biotinylation by BirA*. ( D ) The Venn diagram shows the number of NTHL1-BirA* target proteins identified from mass spectrometry after removing all candidates with BFDR above 0.2, and proteins identified in cells expressing BirA*-FLAG and NLS-BirA*-FLAG. 146 preys were identified in both unirradiated and irradiated cells, while 41 and 36 preys were respectively identified only in unirradiated or irradiated cells. ( E ) Nuclear protein extracts (500 μg) from MDA-MB-231cells were submitted to immunoprecipitation (IP) alternatively with NTHL1 or BCL11A antibodies and then immunoblotted (IB) with either anti-NTHL1 or anti-BCL11A antibody. The control lanes did not include a primary antibody while the input was loaded directly onto the gel. ( F ) 293 cells were transfected with vectors expressing BCL11A and NTHL1 fusion proteins containing the N-terminal or C-terminal portion of intein, respectively: BCL11A-V5-IN and IC2-FLAG-NTHL1. Whole cell extracts were submitted to immunoblotting analysis with the V5 and FLAG antibodies. The new band detected by both antibodies represents the recombined protein.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Lysis, Affinity Purification, Mass Spectrometry, Western Blot, Stable Transfection, Expressing, Irradiation, Immunoprecipitation, Transfection

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A knockdown causes an increase in genomic DNA damage and a delay in the repair of oxidized bases and abasic sites. MDA-MB-231 cells were infected with lentiviral vectors expressing a BCL11A shRNA or a non-targeting sequence. Knockdown of BCL11A is ∼60%. ( A ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated times before carrying out single cell gel electrophoresis at pH > 13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( B ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH 10 and pH 10 after treatment of cells with the Endo III DNA glycosylase. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) Cells stably carrying a lentivirus expressing an shRNA against BCL11A or an empty vector were treated or not with 50 μM H 2 O 2 . Genomic DNA was purified and abasic sites were quantified using an aldehyde-reactive probe. Results are an average of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Infection, Expressing, shRNA, Sequencing, Single Cell Gel Electrophoresis, Stable Transfection, Plasmid Preparation, Purification

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Ectopic Expression of BCL11A 160–520 Accelerates DNA Repair and Increases Resistance to H 2 O 2 . ( A ) RPE1 cells expressing the BCL11A 160–520 peptide were exposed to 100 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( B ) RPE1 cells expressing the BCL11A 160–520 peptide were treated with 0, 50 or 100 μM H 2 O 2 and submitted to a clonogenic assay. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Expressing, Single Cell Gel Electrophoresis, Clonogenic Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A cooperates with RAS to transform primary cells and escape senescence. IMR90 primary fibroblastic cells were infected with retroviruses expressing either HRAS alone or HRAS and BCL11A 160–520 , as indicated. ( A ) Total cell extracts were analyzed by immunoblotting with the indicated antibodies. ( B ) Cells were plated in soft agar 2 days after infection and colonies were counted after 2 weeks Results are an average of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) IMR90 cells were selected for 5 days with puromycin, cells were then submitted to single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( D ) β-Gal associated senescence was measured 7 days after infection with vectors expressing HRAS or HRAS and BCL11A 160–520 . Values were normalized to the value of empty vector. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( E ) Expression of the senescence markers P16, P21, IL6 and IL8 was measured by RT-PCR 5 days following infection of IMR90 cells with vectors expressing either HRAS, BCL11A 160–520 , HRAS and BCL11A 160–520 or nothing. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Infection, Expressing, Western Blot, Single Cell Gel Electrophoresis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Genes

Article Title: Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

doi: 10.3390/genes11020120

Figure Lengend Snippet: Effects of NR2F2 knockdown on progesterone biosynthesis. ( A ) gene expression of NR2F2 after siRNA transfection; ( B ) Effects of NR2F2 knockdown on progesterone secretion; ( C ) gene expression of key factors (StAR, Cyp11a1, HSD3B, and NPC1) involved in progesterone biosynthesis; ( D , E ) protein expression of NR5A2, StAR, Cyp11a1, and HSD3B. Primary pGCs were transfected with NR2F2 siRNA (siNR2F2) or scramble siRNA (siNC). Six hours later, the medium was changed to fresh medium with DMSO or DAPT, followed by further culture for 48 h. * means significant difference at p < 0.05.

Article Snippet: The proteins were subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany) and probed with primary antibodies against StAR (DF6192, Affinity Biosciences, Changzhou, China), Cyp11a1 (DF6569, Affinity Biosciences), HSD3B (DF6639, Affinity Biosciences), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), pERK1/2 (4370T, Cell Signaling), NR2F2 (NBP1-31980, Novus Biologicals, CO, USA), NR5A2 (NBP2-27196SS, Novus) and β-actin (20536-1-AP, Protein Tech, Wuhan, China).

Techniques: Knockdown, Gene Expression, Transfection, Expressing

Journal: Genes

Article Title: Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

doi: 10.3390/genes11020120

Figure Lengend Snippet: Effects of NR5A2 and NR2F2 knockdown on the expression of Notch receptors and ligands. ( A ) Effects of NR5A2 and NR2F2 on the expression of Notch receptors (Notch2 and Notch3) and Notch ligands (Dll1 and Jagged1). Primary pGCs were transfected with NR5A2 siRNA (siNR5A2), NR2F2 siRNA (siNR2F2), or scramble siRNA (siNC). ( B ) Schematic representation of the Notch/NR5A2/NR2F2 pathway in regulating steroidogenesis pGCs. * means significant difference at p < 0.05.

Article Snippet: The proteins were subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany) and probed with primary antibodies against StAR (DF6192, Affinity Biosciences, Changzhou, China), Cyp11a1 (DF6569, Affinity Biosciences), HSD3B (DF6639, Affinity Biosciences), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), pERK1/2 (4370T, Cell Signaling), NR2F2 (NBP1-31980, Novus Biologicals, CO, USA), NR5A2 (NBP2-27196SS, Novus) and β-actin (20536-1-AP, Protein Tech, Wuhan, China).

Techniques: Knockdown, Expressing, Transfection

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human anti-CD142 (F3)-APC , Milteny , 130-115-685.

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software