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: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 6. Non-homologous DNA end joining (NHEJ) pathway. A, NHEJ assay. Ten micrograms of nuclear lysate from lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) was added to 250 ng of Sma I–digested (SmaI[]) pUC19 DNA. Nuclear lysates from all SLE cell lines and a wild-type (WT) control cell line (Paris1) efficiently ligated Sma I–digested plasmid, forming higher-order multimers (upper right). Exclusion of MgCl2 (WT[]MgCl) served as a negative control (see Materials and Methods). Quantification of ligation efficiency was calculated using the dimer and a representative multimer band (asterisk). A linear form of the plasmid (Uncut pUC19) was used as the loading control. B, Immunoblot analysis of 7 core proteins involved in NHEJ. Fifty micrograms of whole cell extract isolated from each SLE lymphoblastoid cell line was used to immunoblot for DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), Ku70, Ku80, Artemis, XLF/Cernunnos (XLF), DNA ligase IV (Lig 4), and XRCC4. Beta-actin or structural maintenance of chromosomes protein 1 (SMC1) was used as a loading control and is shown below the corresponding immunoblots. All 7 core proteins were present in the 16 SLE cell line extracts tested. SLE 64 and SLE 73 were retested for Ku70 and Ku80; SLE 68 was retested for XRCC4 and Lig 4. All cell lines were normal (not all data are shown).

Article Snippet: Blots were incubated for 2 hours at room temperature or overnight at 4°C with antibodies to SMC1, Artemis, Lig 4, XLF/Cernunnos, and XRCC4 (all from Novus Biologicals), Ku70/80 (GenTex), and DNA-PKcs and -actin (both from Abcam).

Techniques: NHEJ Assay, Control, Plasmid Preparation, Negative Control, Ligation, Western Blot, Isolation

Journal: Bioanalysis

Article Title: Quantification of XRCC and DNA-PK proteins in cancer cell lines and human tumors by LC-MS/MS.

doi: 10.4155/bio.14.121

Figure Lengend Snippet: Figure 1. Extract ion chromatogram of digested peptides from XRCC4 protein in bovine serum albumin. Extracted ion chromatograms are shown for five peptides from XRCC4 (90 fmol on column) spiked into 100 ng/µl of bovine serum albumin (500 ng on column). Each extracted ion chromatogram represents one selected fragment ion from the targeted XRCC4 peptide precursor ion.

Article Snippet: Materials & reagents The unlabeled and heavy isotope-labeled XRCC4 proteins were purchased from Origene (MD, USA).

Techniques:

Journal: Bioanalysis

Article Title: Quantification of XRCC and DNA-PK proteins in cancer cell lines and human tumors by LC-MS/MS.

doi: 10.4155/bio.14.121

Figure Lengend Snippet: Figure 2. MS/MS spectra of XRCC4 peptide ALLSGAGPADVYTFNFSK (929.4727, 2+) from QC in bovine serum albumin. (A) Chromatogram and (B) MS/MS of the ALLSGAGPADVYTFNFSK peptide from light XRCC4 (90 fmol on column) spiked into bovine serum albumin 500 ng on column. (C) Standard curve for digested XRCC4 peptide ALLSGAGPADVYTFNFSK; displays the curve and r2 value for the ALLSGAGPADVYTFNFSK peptide spiked into bovine serum albumin. The curve is comprised of seven points where heavy-labeled XRCC4 is spiked in at a constant concentration of 9 fmol/µl with increasing concentration spikes of light XRCC4. The smaller insert displays the lower curve with five lower standards (0.028, 0.14, 0.7, 3.5 and 18 fmol).

Article Snippet: Materials & reagents The unlabeled and heavy isotope-labeled XRCC4 proteins were purchased from Origene (MD, USA).

Techniques: Tandem Mass Spectroscopy, Labeling, Concentration Assay

Journal: Bioanalysis

Article Title: Quantification of XRCC and DNA-PK proteins in cancer cell lines and human tumors by LC-MS/MS.

doi: 10.4155/bio.14.121

Figure Lengend Snippet: Figure 3. MS/MS spectra of XRCC4 peptide ALLSGAGPADVYTFNFSK (929.4727, 2+) from HEK‑293 cell line. (A) Chromatogram and (B) MS/MS of the ALLSGAGPADVYTFNFSK peptide from the XRCC4 protein detected in HEK‑293 cells (500 ng total proteins on column).

Article Snippet: Materials & reagents The unlabeled and heavy isotope-labeled XRCC4 proteins were purchased from Origene (MD, USA).

Techniques: Tandem Mass Spectroscopy