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