Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A ) ParC5 shNT and shδ110 NHEJ reporter cells were generated that stably express shSCR, shSIRT6–1, shSIRT6–2, or shSIRT6–3. Lysates were immunoblotted for the indicated proteins. ( B ) Top: Histone fractions from ParC5 shNT/shδ110-shSCR/shSIRT6–2/shSIRT6–3 cells were acid-extracted and immunoblotted for H3K36me2 and total H3. Bottom: Densitometry of H3K36me2 normalized to total H3. ( C ) MNase assay was performed on ParC5 shNT/shδ110-shSCR/shSIRT6–2/shSIRT6–3 cells. Percentage of DNA in each [NaCl] fraction was assayed as described in . ( D ) DNA repair was analyzed in ParC5 shNT/shδ110-shSCR/shSIRT6–2/shSIRT6–3 NHEJ reporter cells and quantified as described in . ( E to H ) Lysates from ParC5 shNT/shδ110-shSCR/shSIRT6–2/shSIRT6–3 cells were immunoblotted with the indicated antibodies. Densitometry of DNA Lig4 (F), XRCC4 (G), and XLF (H) normalized to their corresponding β-actin control from the Western blots shown in (E). ( I ) ParC5 shNT/shδ110-shSCR/shSIRT6–2/shSIRT6–3 cells were IR at 10 Gy and allowed to recover for 48 hrs. Cells were harvested and caspase-3 activity was assayed. Shown is the fold induction of caspase-3 activities in each cell line over their own corresponding no IR controls. Shown is data from at least three independent biological replicates. Data is shown as mean ± SEM. Statistics represent two-way ANOVA followed by Tukey’s multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Antibodies for DNA Ligase IV (Cat # sc-271299, RRID:AB_10610371), XRCC4 (Cat # sc-271087, RRID:AB_10612396), XLF (Cat # sc-166488, RRID:AB_2152940), SIRT6 (Cat # sc-517556, RRID:AB_2915920), and Mre11 (Cat # sc-135992, RRID:AB_2145244) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Generated, Stable Transfection, Control, Western Blot, Activity Assay

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: Frontiers in Cell and Developmental Biology

Article Title: IGF1R promotes radiation-induced HSCs activation by regulating DNA-PKcs-mediated DNA damage repair

doi: 10.3389/fcell.2025.1678654

Figure Lengend Snippet: Role of IGF1R in promotion of IR-induced HSCs activation mediated through the DNA damage repair process activated by DNA-PKcs. (A) Level of DNA damage foci of γ-H2AX in LX-2 and HSC-T6 cells in the NC and OE groups after co-irradiation with 6 Gy or 8 Gy, detected 3 h after IR. The cell nuclei were counterstained with DAPI (blue), and γ-H2AX was stained green, measured by IF. (B) DNA damage in LX-2 and HSC-T6 cells, measured using the alkaline comet assay; the length of the comet tail represents the severity of DNA damage. (C) Results of correlation analysis of IGF1R with fibrosis-related genes and DNA damage-related genes in human liver fibrosis samples using Spearman’s correlation coefficient using data from the GEO database. (D) The DNA-PKcs expression level detection in the DNA damage time point of the HSC-T6 cell activation process induced by irradiation at 0.5 h, 1 h, 3 h, and 6 h after IR. (E) The levels of p-IGF1R, IGF1R, XRCC4, and XLF in LX-2 and HSC-T6 cells in the OE and sh-IGF1R groups compared to those in the NC and sh-NC groups, respectively, or after co-irradiation with 6 Gy or 8 Gy detected 3 h after IR. (F) The quantitative analysis of the levels of p-IGF1R, IGF1R, XRCC4, and XLF in LX-2 and HSC-T6 cells in the OE and sh-IGF1R groups compared to those in the NC and sh-NC groups, respectively, or after co-irradiation with 6 Gy or 8 Gy.

Article Snippet: The blocked PVDF membrane, which transferred total proteins, was incubated overnight at 4 °C with primary antibodies specific to IGF1R (CST, United States), DNA-PKcs (CST, United States), α-SMA (Proteintech, China), collagen-1 (Proteintech, China), XRCC4 (Proteintech, China), XLF (Proteintech, China), GAPDH (Proteintech, China), and HRP-conjugated secondary antibody (BBI, China) for 1 h at room temperature.

Techniques: Activation Assay, Irradiation, Staining, Alkaline Single Cell Gel Electrophoresis, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: IGF1R promotes radiation-induced HSCs activation by regulating DNA-PKcs-mediated DNA damage repair

doi: 10.3389/fcell.2025.1678654

Figure Lengend Snippet: Role of IGF1R in promoting IR-induced HSCs activation, mediated through the DNA damage repair process activated by DNA-PKcs. (A) Levels of α-SMA and collagen-1 in LX-2 and HSC-T6 cells treated with the DNA-PKcs inhibitor, respectively, or after co-irradiation. (B) Quantitative analysis of the levels of α-SMA and collagen-1 in LX-2 and HSC-T6 cells treated with the DNA-PKcs inhibitor, respectively, or after co-irradiation. (C) The levels of p-IGF1R, IGF1R, XRCC4, XLF, α-SMA, and collagen-1 in LX-2 cells in the OE and NC groups treated with the DNA-PKcs inhibitor, respectively, or after co-irradiation with 6 Gy.

Article Snippet: The blocked PVDF membrane, which transferred total proteins, was incubated overnight at 4 °C with primary antibodies specific to IGF1R (CST, United States), DNA-PKcs (CST, United States), α-SMA (Proteintech, China), collagen-1 (Proteintech, China), XRCC4 (Proteintech, China), XLF (Proteintech, China), GAPDH (Proteintech, China), and HRP-conjugated secondary antibody (BBI, China) for 1 h at room temperature.

Techniques: Activation Assay, Irradiation

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