Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes a transcriptional downregulation of RAD51 and other proteins known to control DSB repair by HR. RNA-seq was performed on U2OS cells transfected with non-silencing (NS), NFKB2 or RELB siRNA. ( A ) Knockdown is efficient for both p100/52 and RelB, based on mRNA measurements by RNA-seq (left) and protein level by western blot (right). ( B ) KEGG pathway analysis of was performed to predict the 10 most upregulated and 10 most downregulated pathways in response to siNFKB2. Corresponding effects in response to siRELB are also displayed. ( C ) Expression levels are displayed for 39 genes with known relevance to HR, as defined by the KEGG database. Genes are arrayed from left to right based on degree of expression change after siNFKB2 (* denotes P < 0.01).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Control, RNA Sequencing, Transfection, Knockdown, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes downregulation of RAD51 protein. ( A ) Representative western blots demonstrate that transcriptional silencing of key NF-κB proteins leads to reduced levels of RAD51 protein. ( B ) Quantitation of western blots from three independent experiments demonstrate that RAD51 reductions after siNFKB2 are reproducible (error bars denote the SEM). ( C ) Comparable levels of RAD51 knockdown are observed using three different siRNAs (each at 25 nM) targeting NFKB2 in U2OS cells. ( D ) Forced overexpression of the p52 fragment of Nfkb2 in mouse lung tissue is associated with increased Rad51 expression (error bars denote standard error, * denotes P < 0.02, NS denotes non-silencing control).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Western Blot, Quantitation Assay, Knockdown, Over Expression, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 inhibits DNA damage-induced RAD51 focus formation in human cancer cells. Cells previously transfected with siRNA or non-silencing (NS) controls were pulse-labeled with EdU to mark S-phase cells and concomitantly treated with 30 nM CPT for 1 hour. Cells were harvested and immune-stained as indicated. Representative microscopic images of unselected nuclei ( A ) are displayed for cells collected 3-hours after CPT treatment, and ( B ) quantitation of RAD51 foci per nucleus is plotted for each of the indicated conditions. ( C ) Quantitation of EdU intensity in CPT-untreated cells is used to estimate cell cycle at the time of the EdU pulse. ( D ) A schematic representation of the treatment timeline is displayed. ( E ) The mean number of RAD51 foci per EdU-positive nucleus is plotted as a function of time following CPT treatment (error bars denote SEM for at least 100 nuclei within a single experimental replicate, * denotes P < 0.05, *** denotes P < 1e−13, and ns denotes P > 0.05).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Transfection, Labeling, Staining, Quantitation Assay

Journal: Stem Cell Reports

Article Title: Endogenous DNA Damage Leads to p53-Independent Deficits in Replicative Fitness in Fetal Murine Fancd2 − / − Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.stemcr.2016.09.005

Figure Lengend Snippet: DNA-Damage Responses Are Induced, and the Strand Breaks Are Accumulated in FA FL HSPCs (A) Representative γH2AX foci in SCA1 + FL cells (60× objective lens; the scale bar represents 5 μm). (B) Quantification of γH2AX foci per cell (n = 3 WT and 3 KO from three litters). (C) Representative RAD51 foci in SCA1 + FL (60× objective lens; the scale bar represents 5 μm). (D) Quantification of cells positive for foci (p = 0.02, n = 6 WT and 6 KO from three litters). (E) Expression of DDR genes in SCA1 + FL cells (p = 0.08, 0.44, 0.06, 0.22, and 0.005, respectively; n = 4 WT and 4 KO from three litters). (F) Expression of selected DDR genes in ASL-sorted FL cells (p = 0.08, 0.5, 0.04 respectively; n = 3 WT and 3 KO from two litters). (G) Olive tail moment of 428 SCA1 + FL cells from seven WT animals and 288 SCA1 + FL cells from five Fancc − / − animals from four litters (p = 0.003). (H) Olive tail moment of 289 SCA1 + FL cells from five WT animals and 267 SCA1 + FL cells from five Fancd2 − / − animals from four litters (p = 0.04). (I) Representative alkaline comets of SCA1 + FL cells (20× objective lens; the scale bar represents 20 μm). Error bars reflect SEM, and asterisks indicate ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01.

Article Snippet: Cells were permeabilized with NET buffer with 0.5% Triton X-100 ( ) and stained with Hoechst (Thermo Scientific 62249) and AF488-conjugated anti-γH2AX (BioLegend, 613407), rabbit anti-mouse RAD51 (Santa Cruz Biotechnology, 8349) followed by Cy3 goat anti-rabbit IgG (Millipore, AP132C) or Cy5-conjugated anti-phospho-p38 (T180/Y182) (eBioscience, 17-9078-42).

Techniques: Expressing

Journal: Cell reports

Article Title: Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia

doi: 10.1016/j.celrep.2023.112027

Figure Lengend Snippet: (A and B) Immunofluorescence of AML cell lines. (A) Representative images of MOLM-13 and THP-1 cells with γH2AX, RAD51, and DAPI after 72-h treatment with vehicle, LAA (250 μM), Olap (1 μM), or LAA + Olap. Scale bar, 25 μm. (B) γH2AX (top) and RAD51 (bottom) focus/cell quantitation. (C) Relative γH2AX (top) and RAD51 (bottom) MFI in MOLM-13 and THP-1 cells treated with LAA and Olap for 72 h. (D–F) Cell cycle dynamics of LAA + Olap-treated cells. (D) Representative density plots of MOLM-13 and THP-1 cells treated for 72 h with vehicle, LAA, Olap (Olap), or LAA + Olap and stained for EdU incorporation and DAPI. (E and F) Relative frequency of MOLM-13 (top) and THP-1 (bottom) cells (E) and γH2AX MFI within each cell cycle gate (F), normalized to vehicle. Mean ± SEM of 3 or more experiments. *p < 0.05, **p < 0.005 (normalized to vehicle), #p < 0.05 (normalized to Olap). (G and H) Cellular proliferation and cycling dynamics. (G) Schematic of BrdU pulse pre-treatment, followed by 72-h treatment with vehicle, LAA, Olap, or LAA + Olap, and EdU chase. (H) Relative frequency of BrdU + (left) and percentage of EdU + BrdU + (right) of THP-1 cells. Representative experiment, mean ± SEM. (J) MOLM-13 and THP-1 cells treated for 72 h with vehicle, LAA, Olap, or LAA + Olap, stained for Annexin V and DAPI, live (double negative), early apoptotic (Annexin V + DAPI − ), or late apoptotic (Annexin V + DAPI + ). Representative experiment, mean + SD. (I) Comet assay performed on MOLM-13 and THP-1 cells treated for 72 h with vehicle, LAA, Olap, or LAA + Olap or for 2 h with 20 μM etoposide. Shown is the tail moment of treated THP-1 (left) and representative fluorescence images of treated MOLM-13 and THP-1 cells (right). Scale bar, 200 μm. (K and L) Quantification of MOLM-13 and THP-1 cells (seeded at 0.2 × 10 6 cells/mL) (K) and relative p21 MFI (L) upon treatment with vehicle, LAA, Olap, or LAA + Olap for 72 h. Mean ± SEM, n = 2 experiments. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005.

Article Snippet: anti-human RAD51 antibody , B-Bridge International , Cat# 70-001; RRID:AB_2177110.

Techniques: Immunofluorescence, Quantitation Assay, Staining, Single Cell Gel Electrophoresis, Fluorescence

Journal: Cell reports

Article Title: Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia

doi: 10.1016/j.celrep.2023.112027

Figure Lengend Snippet:

Article Snippet: anti-human RAD51 antibody , B-Bridge International , Cat# 70-001; RRID:AB_2177110.

Techniques: Purification, Blocking Assay, Recombinant, Lysis, Staining, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Binding Assay, Single Cell Gel Electrophoresis, Software, Protein Array

Journal: International Journal of Medical Sciences

Article Title: IER5 is involved in DNA Double-Strand Breaks Repair in Association with PAPR1 in Hela Cells

doi: 10.7150/ijms.21510

Figure Lengend Snippet: IER5 regulates NHEJ-mediated DSB repair. a. Western blot indicating IER5 and 53BP1 knockdown by specific siRNAs. b. FACS analyses of NHEJ assay in Hela cells treated with IER5 and 53BP1 siRNAs. c. Quantification of the NHEJ assay. d. Western blot indicating IER5 and RAD51 knockdown in Hela cells upon treatment with specific siRNAs. e. Efficiency of HR, as analyzed by FACS. f. Quantification of the HR assay.

Article Snippet: Antibodies used in this study: anti-IER5 goat polyclonal antibody (Abcam), anti-IER5 rabbit polyclonal antibody (Santa Cruz), anti-GAPDH mouse monoclonal antibody (Zhong Shan Jin Qiao), anti-PARP1 rabbit monoclonal antibody (Santa Cruz), anti-Ku70 mouse monoclonal antibody (Abcam), anti-Ku80 rabbit monoclonal antibody (Santa Cruz), anti-FLAG M2 mouse monoclonal antibody (Sigma), anti-53BP1 rabbit polyclonal antibody (Santa Cruz), anti-RAD51 rabbit polyclonal antibody (Proteintech), anti-γH2AX mouse monoclonal antibody (Millipore), anti-Ubiquitinylated proteins mouse monoclonal antibody (Millipore), and anti-PADPR mouse monoclonal antibody (Abcam).

Techniques: Western Blot, Knockdown, NHEJ Assay