Journal: Molecular Cancer Therapeutics

Article Title: Functional Genetic Screen Identifies Increased Sensitivity to WEE1 Inhibition in Cells with Defects in Fanconi Anemia and HR Pathways

doi: 10.1158/1535-7163.mct-14-0845

Figure Lengend Snippet: Figure 2. WEE1 inhibition induces replication stress and premature mitosis. A, immunofluorescence of gH2AX (green) in WiDr cells treated with 250 nmol/L MK-1775 for the indicated times. DNA was counterstained with DAPI (blue). Representative images and box and whisker plot of gH2AX intensity/cell are shown. Pan-nuclear gH2AX staining indicates widespread replication fork stalling. B, time course of 53BP1 (red) and gH2AX (green) immunofluorescence after WEE1 inhibition (250 nmol/L). Representative images and average number of 53BP1 foci per cell of two independent experiments are shown. C, flow- cytometric analysis of cells stained for gH2AX (left) and mitotic marker phospho-histone H3 (pH3; right) after treatment with 200 nmol/L MK-1775 for the indicated times. Left panel illustrates two distinct populations of cells positive for gH2AX (green, in boxed area): gH2AXhigh and gH2AXlow; and a back-gated population of pH3-positive cells that is gH2AX-negative (in red). In the right panel, the gH2AXhigh and gH2AXlow cells are back-gated in a pH3 versus DNA content plot (pH3þ cells are gated in the boxed area). The gH2AXhighpH3þ double-positive cells in these plots are premature mitotic (green cells in boxed area), whereas gH2AXlow cells were mainly in S or G2 phase (green cells outside boxed area). Normal mitotic cells positive for pH3 but not gH2AX are shown in red. D, intracellular dTTP levels were determined in cell extracts from WiDr cells treated with MK-1775 for 2 hours (black bars) or 4 hours (gray bars). Data were normalized to untreated control cells. E, nucleoside (nucl) supplementation reduced the number and intensity of gH2AX- positive cells after WEE1 inhibition. Representative immunofluorescence images and a box and whisker plot of gH2AX intensity/cell are shown of cells treated with 250 nmol/L MK-1775 for 8 hours in the absence (white bars) or presence of nucleosides (gray bars). F, quantification of mitotic (pH3þ) and premature mitotic (gH2AXhighpH3þ) cells by flow-cytometric analysis. Cells were treated as described in C. nucl indicates supplementation with nucleosides. For gH2AXhighpH3þ cells: , P < 0.04 versus MK-1775 alone (ratio paired Student t test).

Article Snippet: Antibodies used were phospho-Histone H3-Ser10 (pH3; 06-570, Upstate), phospho-Histone-H2AX-Ser139 (05-636, Upstate; 9718, Cell Signaling Technology), b-actin (A5441), b-tubulin (T4026, both from Sigma-Aldrich), CHK1 (sc-8408, Santa Cruz Biotechnology), MYT1 (4282), MRE11 (4895), Ezrin (3145; all from Cell Signaling Technology), BRIP1 (NB100-416), FANCD2 (NB100-182), and 53BP1 (NB100304; all from Novus Biologicals). siRNA screening Screening was performed in 384-well plates with a Dharmacon siGENOME SMARTpools library targeting all known protein kinases, phosphatases, tumor suppressor, and DNA repair genes.

Techniques: Inhibition, Whisker Assay, Staining, Marker, Control

Journal: The Journal of cell biology

Article Title: The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks.

doi: 10.1083/jcb.201205059

Figure Lengend Snippet: Figure 4. DNA damage signaling is not af- fected by p400 knockdown. (A and B) U2OS cells transfected with control or p400 siRNA were irradiated (8 Gy) or not 48 h later and subjected to FK2 immunofluorescence. Repre- sentative images (A) and quantifications are shown (B). Bar, 10 µm. Results are the mean ± SD from three independent experiments. (C and F) Automatized analysis of ubiquitin (FK2; C) or 53BP1 (F) foci in U2OS cells ex- posed to 8 Gy. Note that in other experiments the decrease was less pronounced after the 30-min peak of FK2 foci. (D and G) Automa- tized analysis of ubiquitin (FK2; D) or 53BP1 (G) foci in U2OS cells exposed to 2 Gy. (E and H) Automatized analysis of ubiquitin (FK2; E) or 53BP1 (H) foci in 293T cells exposed to 8 Gy. For C–H, the data shown are from a single representative experiment (n = 200) out of three repeats.

Article Snippet: The commercial p400 antibody was purchased from Abcam, the Rad51 antibodies from Santa Cruz Biotechnology, Inc. (Rad51-H92; for ChIPs and some Western blot experiments) or from EMD Millipore (for some Western blot experiments), the p21 antibody (C19) from Santa Cruz Biotechnology, Inc., the HA antibody from Covance (HA-11), the myc antibody from Roche (9E10), the H2AX antibodies from EMD Millipore for immunofluorescence experiments (JBW301) and from Epitomics for Western blot experiments, the actin and -tubulin antibodies from Sigma-Aldrich (T6199), the phospho-ATM antibody from Cell Signaling technology (10H11.E12), the lamin A/C antibody from Tebu-Bio (636), the phosphoRPA antibodies from Bethyl Laboratories, Inc. (S4-8) for immunofluorescence and (S33) for Western blot experiments, p-ChK2 antibody from Cell Signaling Technology (T68), BRCA1 antibody from Santa Cruz Biotechnology, Inc. (D-9), the FK2 antibody from EMD Millipore, the Rad52 antibody from Abcam, and the 53BP1 antibody from Novus Biologicals.

Techniques: Knockdown, Transfection, Control, Irradiation, Immunofluorescence, Ubiquitin Proteomics

Journal: Radiation oncology (London, England)

Article Title: Transcriptomic response of prostate cancer cells to carbon ion and photon irradiation with focus on androgen receptor and TP53 signaling.

doi: 10.1186/s13014-024-02480-z

Figure Lengend Snippet: Fig. 2 Analysis of DNA-damage in LNCaP and DU145 cells following photon and 12C-ion irradiation. A Immunofluorescence of γ-H2AX (red), 53BP1 staining (green) and DAPI-merge (weakly blue) for counterstaining of nuclei. The co-localized red and green foci indicate the DNA-double-strand-breaks (DSB) per nucleus. The white bar scale corresponds to 10 µm. The respective images of dose and time served for counting of DSB-foci. B Number of DSB-foci for photon and 12C-ion irradiation of LNCaP (left) and DU145 (right) time dependently for each dose of photon or 12C-ion irradiation (mean ± SD, n ≥ 95). C Western-blot analysis of γ-H2AX and H2AX, after (2 h) photon or 12C-ion irradiation, dose-dependent in LNCaP cells (left) and DU145 cells (right)

Article Snippet: Fixed cells were permeabilized with 0.2% Triton X-100, 1% BSA/ PBS for 10 min, washed with 1% BSA/PBS and blocked in 3% BSA/PBS for 1 h. The primary antibody solution was incubated for 1.5 h at room temperature using the following antibodies: mouse monoclonal anti-phosphoS139-H2AX antibody (1:500, clone JBW301, Millipore, Darmstadt, Germany) and rabbit polyclonal 53BP1 antibody (1:500, Novus Biologicals, Wiesbaden, Germany).

Techniques: Irradiation, Immunofluorescence, Staining, Western Blot