Journal: Journal of Assisted Reproduction and Genetics

Article Title: The DNA double-strand break repair proteins γH2AX, RAD51, BRCA1, RPA70, KU80, and XRCC4 exhibit follicle-specific expression differences in the postnatal mouse ovaries from early to older ages

doi: 10.1007/s10815-024-03189-4

Figure Lengend Snippet: Cellular distribution and relative levels of XRCC4 protein in the postnatal mouse ovaries. a Representative microscopic micrographs of XRCC4 immunostaining of prepuberty (PreP, n = 6), puberty (P, n = 7), postpuberty (PostP, n = 7), early aged (EA, n = 7), and late aged (LA, n = 7) groups. XRCC4 protein was intensely expressed in oocytes and granulosa cells of the follicles at different developmental stages as well as in granulosa lutein and theca lutein cells. The asterisks indicate small spaces between granulosa cells, the red arrows show germinal epithelium. The micrographs and their inserts were captured at 200 × and 400 × original magnifications, respectively. Scale bars represent 50 µm. O, Oocyte; TL, Theca layer; S, Stroma; SF, Secondary follicle; PAF, Preantral follicle; AF, Antral follicle; AtF, Atretic follicle; CL, Corpus luteum. b The relative XRCC4 protein levels in the total area. It gradually increased from the prepuberty to the late aged groups ( P < 0.05). c-h The relative XRCC4 protein levels in c primordial, d primary, e secondary, f preantral, g antral, and h atretic follicles, and in oocyte’s cytoplasm (C) and nucleus (N), granulosa cells, and theca cells. Although we found no changes in primordial and atretic follicles, there was an increasing trend of XRCC4 expression in primary, secondary, preantral, and antral follicles toward to the early or late aged group when compared to the early groups ( P < 0.05). i The relative XRCC4 protein levels in ovarian cells located in the germinal epithelium, stroma, and corpus luteum. It increased in stromal cells, granulosa lutein and theca lutein cells from the prepuberty to the late aged groups ( P < 0.01). Data were analyzed using one-way ANOVA and Tukey’s post hoc test and are presented as the mean ± standard deviation (SD). Asterisks above the columns indicate significant differences as follows: * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Next, the sections were incubated with γH2AX (diluted 1:1000; catalog no. 9718S, Cell Signaling Technology, Danvers, MA, USA), RAD51 (diluted 1:250; catalog no. bs-20297R, Bioss Inc., Woburn, MA, USA), BRCA1 (diluted 1:1000; catalog no. bs-0803R, Bioss Inc.), and RPA70 (diluted 1:3000; catalog no. 2267S, Cell Signaling Technology), KU80 (diluted 1:250; catalog no. MBS712948, MyBiosource, Inc., San Diego, CA, USA), XRCC4 (diluted 1: 250; catalog no. bs8510R, Bioss Inc.), cCASP3 (diluted 1:300; catalog no. 9661S, Cell Signaling Technology), β-GAL (diluted 1:1300; catalog no. 11518–1-AP, Proteintech Group, Rosemont, IL, USA) primary antibodies at + 4 °C overnight.

Techniques: Immunostaining, Expressing, Standard Deviation

Journal: Cells

Article Title: Cistanoside F Ameliorates Lipid Accumulation and Enhances Myogenic Differentiation via AMPK-Dependent Signaling in C2C12 Myotubes

doi: 10.3390/cells14120874

Figure Lengend Snippet: Effects of Cis on lipid content in C2C12 cells. ( A ) Lipid droplet content of two lipid differentiation models of C2C12 cells, measured using oil red staining and light microscopy. ( B ) C2C12 intracellular lipid drop content detected using TG, TCHO, LDL, and HDL detection kits. ( C , D ) Lipid droplets in two models of significant time of lipid differentiation were measured by a high-content instrument. ( E , F ) CPT1b, PPARγ, ATGL, and UCP1 expression in the IB+R+IN+D model on day 8. n = 6. ## p < 0.01, compared with the vehicle group; * p < 0.05, ** p < 0.01, compared with the model group. ns, no significance.

Article Snippet: Antibodies against UCP1, CPT1b, ACC2, Desmin, and PGC-1α were purchased from Boster Biotech (Wuhan, China; #M00255; #A03558; #A03668; #PB9105; #BM4898; 1:1000).

Techniques: Staining, Light Microscopy, Expressing

Journal: Oncotarget

Article Title: Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody

doi: 10.18632/oncotarget.9003

Figure Lengend Snippet: ( A ) Representative bright field micrographs of a single BJ fibroblast progressing through cytokinesis in the presence (adhesion) and the absence (suspension) of fibronectin contacts, respectively. ( B ) Mean % ± SD of the number of mitotic BJ fibroblasts that completed cytokinesis during the specified time periods after the formation of cleavage furrow. ( C ) Representative fluorescence images illustrating an early and a late cytokinetic stage of BJ cells. The status is based on fluorescence signal distribution of Aurora B as indicator of cleavage furrow width and the presence of CEP55 in the cleavage furrow region. Nuclei were stained with DAPI (blue). ( D ) Mean % ± SD of the number of mitotic BJ cells in the different stages of cytokinesis after the indicated incubation time periods. ( E ) Representative bright field micrographs showing the progression of the cleavage furrow ingression in the mitotic BJ cells adhering to fibronectin or kept in suspension for the indicated time periods. The bars mark the distance between the opposite membrane borders. ( F ) Mean ± SD of the cleavage furrow width. The square frames show the midbody region at higher magnification. *** P -value less than 0.001.

Article Snippet: Antibodies directed against the following proteins were used: Aurora B (ab-2254, rabbit polyclonal and ab-3609 mouse monoclonal, Abcam, Cambridge, UK), CEP55 (sc-134622, rabbit polyclonal and sc-37405, mouse monoclonal, Santa Cruz, California, USA), PLK1 (GTX-104302, rabbit polyclonal, GENTEX, Zeeland, USA), ALIX (3A9, mouse monoclonal, Thermo Fisher scientific, Waltham, USA), TSG101 (GTX-118763, rabbit polyclonal, GENTEX,), α-tubulin (T6199, mouse monoclonal, Sigma, Saint Louis, USA), MKLP1 (sc-22793, rabbit polyclonal, Santa Cruz), CHMP4B (sc-82556, rabbit polyclonal, Santa Cruz), Spastin Sp 3G11/1 (sc-53443, mouse monoclonal, Santa Cruz).

Techniques: Suspension, Fluorescence, Staining, Incubation, Membrane

Journal: Oncotarget

Article Title: Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody

doi: 10.18632/oncotarget.9003

Figure Lengend Snippet: ( A , B , F ) Representative immunofluorescence micrographs illustrating the distribution of selected proteins in the F/M region (labeled with Aurora B) at the beginning of cytokinesis and after 60 minutes incubation of BJ cells adhering to fibronectin or kept in suspension. (A) MKLP1 (green, upper left panel) and CEP55 (green, upper right panel), (B) TSG101 (green, lower left panel) and ALIX (red, lower right panel), (F) CHMP4B (green). The square frames show the midbody area at higher magnification. Arrows indicate midbody regions in the merged pictures. Nuclei were stained with DAPI (blue). Mean % ± SD of the number of mitotic cells in cytokinesis lacking ( C ) MKLP1 and CEP55, ( D ) TSG101 and ALIX and ( G ) CHMP4B in the F/M regions. ***, **, and * represent P -value less than 0.001, 0.01 and 0.05, respectively. ( E ) Representative western blot of cell lysates illustrating the level of ALIX and TSG101 in exponentially proliferating cells (Control) and synchronized mitotic cells directly after isolation (Zero) and following two hours incubation under the adhesion or suspension conditions.

Article Snippet: Antibodies directed against the following proteins were used: Aurora B (ab-2254, rabbit polyclonal and ab-3609 mouse monoclonal, Abcam, Cambridge, UK), CEP55 (sc-134622, rabbit polyclonal and sc-37405, mouse monoclonal, Santa Cruz, California, USA), PLK1 (GTX-104302, rabbit polyclonal, GENTEX, Zeeland, USA), ALIX (3A9, mouse monoclonal, Thermo Fisher scientific, Waltham, USA), TSG101 (GTX-118763, rabbit polyclonal, GENTEX,), α-tubulin (T6199, mouse monoclonal, Sigma, Saint Louis, USA), MKLP1 (sc-22793, rabbit polyclonal, Santa Cruz), CHMP4B (sc-82556, rabbit polyclonal, Santa Cruz), Spastin Sp 3G11/1 (sc-53443, mouse monoclonal, Santa Cruz).

Techniques: Immunofluorescence, Labeling, Incubation, Suspension, Staining, Western Blot, Control, Isolation

Journal: Oncotarget

Article Title: Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody

doi: 10.18632/oncotarget.9003

Figure Lengend Snippet: ( A ) Representative immunofluorescence images illustrating the presence of PLK1 (green) in the F/M regions stained for Aurora B (red) at the beginning of cytokinesis and after 60 minutes incubation of BJ cells adhering to fibronectin or kept in suspension. Nuclei were stained with DAPI (blue). Red squares indicate midbody areas used for the quantification of PLK1 fluorescence signal intensity. ( B ) Mean % ± SD of the number of mitotic cells in cytokinesis lacking PLK1 fluorescence signal in the F/M regions. * P -value less than 0.05. ( C ) Representative immunofluorescence micrographs showing the variation of PLK1 (green) and CEP55 (red) fluorescence signal intensity in the indicated F/M regions at the beginning of cytokinesis and in the indicated time points after the incubation of BJ cells adhering to fibronectin (upper panel) or kept in suspension (lower panel). The corresponding PLK1 and CEP55 signal intensity profiles along the hatched line in each interest area were drawn in the right panel. ( D , E ) Mean intensity ± SE of the PLK1 (D) and CEP55 (E) fluorescence signal intensity in the marked F/M regions 0, 30, 45 and 60 minutes after incubation of the cells adhering to fibronectin or kept in suspension (the background signal in each cell was subtracted). The fluorescence signal intensity of PLK1 and CEP55 were analyzed in 50 mitotic cells for each time point from each of three independent experiments.

Article Snippet: Antibodies directed against the following proteins were used: Aurora B (ab-2254, rabbit polyclonal and ab-3609 mouse monoclonal, Abcam, Cambridge, UK), CEP55 (sc-134622, rabbit polyclonal and sc-37405, mouse monoclonal, Santa Cruz, California, USA), PLK1 (GTX-104302, rabbit polyclonal, GENTEX, Zeeland, USA), ALIX (3A9, mouse monoclonal, Thermo Fisher scientific, Waltham, USA), TSG101 (GTX-118763, rabbit polyclonal, GENTEX,), α-tubulin (T6199, mouse monoclonal, Sigma, Saint Louis, USA), MKLP1 (sc-22793, rabbit polyclonal, Santa Cruz), CHMP4B (sc-82556, rabbit polyclonal, Santa Cruz), Spastin Sp 3G11/1 (sc-53443, mouse monoclonal, Santa Cruz).

Techniques: Immunofluorescence, Staining, Incubation, Suspension, Fluorescence

Journal: Oncotarget

Article Title: Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody

doi: 10.18632/oncotarget.9003

Figure Lengend Snippet: ( A ) Representative immunofluorescence images showing the PLK1 (green) and CEP55 (red) signals at the midbody of mitotic BJ cells in cytokinesis replated on fibronectin and treated with DMSO, FAK inhibitor (PF) and Src inhibitor (SU) for 1 hour. ( B ) Quantification of PLK1 and CEP55 signals at the midbody of cells shown as mean ± SD. ( C ) The variation in the PLK1 and CEP55 fluorescence signal intensities presented as fold change relative to control values after the treatment of the cells with the indicated inhibitors. ( D ) Representative western blot and quantification analysis (mean ± SD; n = 2) showing the expression level of PLK1 in the mitotic cells treated as described above. ( E ) Representative bright field micrograph illustrating the progression of cytokinesis during the indicated time period in a single BJ fibroblast treated with FAK inhibitor (PF). The arrows mark the uncut intercellular bridge between two daughter cells.

Article Snippet: Antibodies directed against the following proteins were used: Aurora B (ab-2254, rabbit polyclonal and ab-3609 mouse monoclonal, Abcam, Cambridge, UK), CEP55 (sc-134622, rabbit polyclonal and sc-37405, mouse monoclonal, Santa Cruz, California, USA), PLK1 (GTX-104302, rabbit polyclonal, GENTEX, Zeeland, USA), ALIX (3A9, mouse monoclonal, Thermo Fisher scientific, Waltham, USA), TSG101 (GTX-118763, rabbit polyclonal, GENTEX,), α-tubulin (T6199, mouse monoclonal, Sigma, Saint Louis, USA), MKLP1 (sc-22793, rabbit polyclonal, Santa Cruz), CHMP4B (sc-82556, rabbit polyclonal, Santa Cruz), Spastin Sp 3G11/1 (sc-53443, mouse monoclonal, Santa Cruz).

Techniques: Immunofluorescence, Fluorescence, Control, Western Blot, Expressing

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: Generation of XRCC4 +/−, XRCC4−/−, and XLF−/−:XRCC4−/− HCT116 cell lines.(A) A diagram representing a partial XRCC4 genomic locus in the HCT116 cell line. Exons are shown (not to scale) as red (5’ and 3’ untranslated regions) or green (coding sequence) colored rectangles. (B) A diagram of the XRCC4 exon 4 Neo rAAV targeting vector. The left and right homology arms (Left HA and Right HA, respectively) used for facilitating HDR between the targeting vector and the genomic locus are shown as blue rectangles. Open and red rectangles depict vector sequences, while LoxP sites are shown as yellow triangles. The orange rectangle represents the Neo gene. The direction and approximate location of vector-specific PCR primers are shown as horizontal arrows. (C) A diagram of a first-round targeted allele using the rAAV-XRCC4 exon 4 Neo targeting vector. The targeting vector has replaced exon 4 on one chromosome. All symbols are the same as in (A and B). Chromosome-specific primers used for determining targeting events are shown as horizontal arrows. (D) A diagram of a XRCC4 exon 4 first-round targeted locus following Cre-mediated LoxP recombination. PCR primers flanking the single LoxP site are shown as horizontal arrows. (E) A diagram of the rAAV-XRCC4 exon 4 fusion Neo targeting vector. All symbols are the same as (A, B and C). (F) A diagram of a first-round targeted allele using rAAV-XRCC4 exon 4 fusion Neo targeting vector. The targeting vector has integrated an in-frame (LoxP site-Neo-PolyA-LoxP) cassette within exon 4 on one chromosome. All symbols are the same as in (A and E). (G) A diagram of a XRCC4 exon 4 fusion first-round targeted allele following Cre-mediated LoxP recombination. All symbols are the same as in (A, E and F). (H) PCR confirmation of XRCC4+/−, XRCC4−/−, and XLF−/−:XRCC4−/− cell lines. A final confirmation PCR was performed using genomic DNA isolated from XRCC4+/−, XRCC4−/Neo, XRCC4−/−, XLF−/− :XRCC4 +/−, XLF−/−:XRCC4Neo/−, and XLF−/−:XRCC4Neo/− clones. Genomic DNA from WT and XLF−/− HCT116 cells was used as controls, lanes 1 and 5 respectively. (I) XRCC4−/− cells lack detectable XRCC4 protein. Whole cell extracts were prepared from parental (WT), LigIV−/−, XRCC4+/−, XRCC4Neo/−, XRCC4−/−, and two XRCC4−/− complemented clones (#4 and #5). The extracts were analyzed by immunoblot for XRCC4 as well as LigIV protein with b-catenin and a-tubulin as loading controls. In the LIGIV panel, a non-specific band is marked with an asterisk (*) and the correct band is marked with an arrow. (J) XLF−/− and XRCC4−/− cells lack detectable XLF and XRCC4 protein. Whole cell extracts were prepared from parental (WT), XLF−/−, XRCC4−/−, as well as two XLF−/−:XRCC4Neo/− cell lines. The extracts were analyzed by immunoblot for XRCC4 expression using Ku70 as a loading control and then subsequently probed with an XLF antibody. (K) PAXX−/− cells lack detectable PAXX protein. Whole cell extracts were prepared from parental (WT), XRCC4−/−, XLF−/−, two PAXX−/−, XRCC4−/−:XLF−/−, XRCC4−/−:PAXX−/−, XLF−/−:PAXX−/−, as well as two XRCC4−/−:XLF−/−:PAXX−/− cell lines. The extracts were analyzed by immunoblotting for XRCC4, XLF and PAXX using a-tubulin as a loading control. In the XRCC4 blot, a non-specific band is marked with an asterisk (*) and the correct band is marked with an arrowhead.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: Sequencing, Plasmid Preparation, Isolation, Clone Assay, Western Blot, Expressing

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: XRCC4-deficient HCT116 cells are sensitive to DNA-damaging agents.(A) Etoposide sensitivity. For each cell line, 3000 cells were plated in duplicate and exposed to the indicated levels of etoposide. Surviving cells that grew into colonies of at least 50 cells after ~10 days were scored. The averages (+/− standard deviations) of two experiments are shown. (B) IR sensitivity. For each cell line, either 300, in the case of the WT and XRCC4+/− cells, or 3000, for the XRCC4−/− and XRCC4−/− complemented cells, were plated in duplicate and X irradiated at the indicated doses. Surviving cells that grew into colonies of at least 50 cells after ~ 10 days were scored. The averages (+/− standard deviations) of two experiments are shown.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: Irradiation

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: NHEJ activity in XRCC4−/−, XRCC4−/− complemented, and XLF−/−:XRCC4Neo/− cells.(A) A diagram of the pEGFP-Pem1-Ad2 NHEJ reporter substrate plasmid. The vector contains an EGFP expression cassette that is driven by a CMV promoter and terminated by the SV40 polyA sequence. The 5’ (G) portion of GFP is separated from the 3’ (FP) portion by a 2.4 kb long Pem1 intron interrupted by an Ad2 exon that is flanked by HindIII and I-SceI restriction enzyme recognition sites. Both splice donor (SD) and splice acceptor (SA) sites are also noted. (B) Restriction enzyme recognition sites used for the introduction of DSBs. Digestion of the two HindIII sites produces compatible cohesive ends while digestion of the inverted 18 bp non-palindromic I-SceI sites produces incompatible ends. (C) The Ad2-exon, within the Pem1 intron, is efficiently spliced into the middle of the GFP ORF resulting in an inactivated GFP gene product that leads to GFP-negative cells. HindIII/I-SceI restriction enzyme recognition sites flank each side of the Ad2 exon. The removal of the Ad2 exon by either of these endonucleases results in a linearized plasmid that, upon successful intracellular recircularization, leads to GFP expression that can be quantitated by flow cytometry. (D) The loss of XRCC4, XLF, as well as both XRCC4 and XLF expression reduces end-joining. The indicated cell lines were transiently co-transfected with HindIII (top panels) or I-SceI (lower panels) linearized pEGFP-Pem1-Ad2 plasmid along with a pCherry plasmid. The cells were analyzed by FACS analysis 24 hr post transfection. The total number of both EGFP- and pCherry-positive (upper right-hand quadrant) cells versus all pCherry-positive (upper left and right-hand quadrant) cells was determined. The resulting percentage for each cell line is shown in the upper right-hand quadrant of each plot. (E) Quantitation of the results shown in (D), plotted as relative plasmid rejoining for HindIII (green bars) or I-SceI (red bars). The graph shown is the average (+/− the standard deviation) from five (WT, XRCC4+/−, XRCC4−/−, XRCC4−/− complemented) or two (XLF−/− and XLF−/−:XRCC4−/−) independent experiments.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: Activity Assay, Plasmid Preparation, Expressing, Sequencing, Flow Cytometry, Transfection, Quantitation Assay, Standard Deviation

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: XRCC4−/−, XLF−/−, and XLF−/−:XRCC4−/− cells exhibit an increase in microhomology-mediated DNA repair.(A) Repaired HindIII-linearized plasmids were recovered from the indicated cell lines and propagated in E. coli for the purpose of determining the total (“perfect rejoining”) frequency. For each cell line, twenty colony PCR products produced using GFP-specific primers were then subjected to HindIII restriction enzyme digestion and subjected to gel electrophoresis. Red asterisks indicate those PCR products where perfect joining occurred. (B) XRCC4−/−, XLF−/−, and XLF−/−:XRCC4−/− cells exhibit elevated use of microhomology-mediated repair. An additional ~ 30 plasmids that had not undergone perfect rejoining were analyzed by DNA sequencing. Those results (compiled from Tables S1 and S2) are displayed here with the percent microhomology used (defined by at least 3 nt of microhomology) for during the repair event. The total number of events (n) analyzed for each sample is indicated by the number above the bars.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: Produced, Nucleic Acid Electrophoresis, DNA Sequencing

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: Confirmation of microhomology-mediated end-joining in XRCC4-deficient cells.(A) A microhomology-directed NHEJ (aEJ) biased reporter substrate plasmid has been engineered so that digestion with Eco47III and EcoRV yields a blunt-ended linear substrate with 6-bp repeats (boxes) at each end. Repair of this plasmid by C-NHEJ joining will result in the retention of both repeats while aEJ should yield a single repeat, which conforms to a BstXI restriction enzyme recognition site. (B) Experimental strategy for analysis of repair events produced in transfected cells. The recovered plasmids are subjected to PCR amplification using primers flanking the repair junction. These ~ 180 bp PCR products are then subjected to BstXI restriction enzyme digestion that will yield 120 bp and 60 bp products if successfully cleaved. The restriction fragments are visualized with SYBR Gold following polyacrylamide gel electrophoresis. (C) The results of one such experiment. (D) Two independent experiments including the one shown in (C) were quantitated using ImageJ software and averaged.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: Plasmid Preparation, Produced, Transfection, Amplification, Polyacrylamide Gel Electrophoresis, Software

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: HCT116 cells lacking XRCC4, XLF, and both XRCC4 and XLF exhibit greatly reduced levels of V(D)J recombination.(A) A human V(D)J recombination substrate plasmid diagram extracted from Gauss and Lieber [46]. Three different plasmids are diagrammed. Each plasmid is a dual drug selection vector that includes an Amp (ampicillin resistance) gene as well as a Ptrp (prokaryotic tryptophan promoter) driving expression of a promoter-less chloramphenicol (Cat) resistance gene. Importantly, a phage transcription terminator is represented by “stop” in the three cassettes. This terminator is flanked by 12- and 23-bp V(D)J RSSs arranged in different orientations for each vector (open and closed arrowheads, respectively). Transfection of each V(D)J substrate plasmid along with RAG1 and RAG2 expression plasmids into human cells and subsequent V(D)J recombination will result in the deletion or inversion of the terminator sequence. Plasmids are then recovered from the human cells and propagated in bacteria, where only recombined plasmids confer resistance to both chloramphenicol and ampicillin while nonrecombined plasmids confer resistance only to ampicillin. (B) V(D)J recombination activity table. The results of five separate experiments for parental (WT) HCT116 as well as three separate experiments for XRCC4-, XLF-, and XLF:XRCC4-deficient cells are shown. (C) The relative V(D)J recombination frequencies for each cell line were determined from the average of these three individual experiments. (D) Incidence of nucleotide deletion from the coding ends. Coding joints (compiled from Fig. S3) were examined for the number of bases lost from each coding end in both directions and binned into the indicated 5 nt intervals. The total number of events (n) analyzed for each genotype is indicated.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: Plasmid Preparation, Selection, Expressing, Transfection, Sequencing, Activity Assay

Journal: DNA repair

Article Title: Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

doi: 10.1016/j.dnarep.2019.102738

Figure Lengend Snippet: rAAV-mediated gene targeting frequency in XRCC4-deficient cells.A graph displaying the relative rAAV-mediated gene targeting frequencies for each locus observed for WT and XRCC4−/− cells. For Artemis and HPRT, the data are from single experiments where 81 to 116 drug-resistant colonies were analyzed for each cell line and for each gene. For PIGA, the data represent the average of two independent cell-sorting experiments that were combined together.

Article Snippet: Antibodies used included mouse anti-XRCC4 (Origene TA500650), rabbit anti-XLF, (Abcam ab126353), rabbit anti-LIGIV, mouse anti-β-catenin (Santa Cruz sc-7963), mouse anti-β-tublin, rabbit anti-PAXX (Abcam, ab126353) and rabbit anti-Ku70 (Santa Cruz sc-9033).

Techniques: FACS