Journal: Journal of Virology

Article Title: Cowpox Virus Expresses a Novel Ankyrin Repeat NF-κB Inhibitor That Controls Inflammatory Cell Influx into Virus-Infected Tissues and Is Critical for Virus Pathogenesis

doi: 10.1128/jvi.00861-09

Figure Lengend Snippet: FIG. 5. vCpx-006KO virus induces the nuclear translocation and transcriptional activity of NF-B. (A) THP1 cells were first differentiated with PMA and then either mock infected or infected with vCpx-gfp or vCpx-006KO virus at an MOI of 3. At 30 min postinfection, cells were harvested, and both cytoplasmic (left) and nuclear (right) extracts were prepared. The levels of NF-B1/p105/p50 and NF-B/p65 were then assessed in each of these extracts using Western blot analysis. As loading controls, levels of -actin and histone H1 were also determined. (B) HeLa cells were cotransfected with a 1:1 mix of plasmids pNFB-Luc (gives inducible NF-B-dependent expression of the firefly luciferase gene) and pRL-TK (gives low-level constitutive expression of the sea pansy luciferase gene). At 24 h posttransfection, cells were either mock infected or infected with untagged CPXV, vCpx-gfp, or vCpx-006KO (at an MOI of 10) for 4 h. Cells were either left untreated or treated with TNF- (20 ng/ml) for 4 h and then assayed for both sea pansy and firefly luciferase activities. The values presented represent the averages of data from three independent transfections. Statistical significance was calculated using the Student t test.

Article Snippet: Rabbit polyclonal antibody for histone H1 was obtained from Santa Cruz Biotechnology, and mouse monoclonal antibody for -actin was obtained from Ambion.

Techniques: Virus, Translocation Assay, Activity Assay, Infection, Western Blot, Expressing, Luciferase, Transfection

Journal: Journal of cell science

Article Title: Forced binding of the origin of replication complex to chromosomal sites in Drosophila S2 cells creates an origin of replication.

doi: 10.1242/jcs.094409

Figure Lengend Snippet: Fig. 6. UAS region shows K12-acetylated H4 when it is used as an origin. ChIP analysis was carried out using antibodies against V5 or acetylated K12. PCR analysis was performed using the oriDa region as the negative control. (A) H4K12 acetylation observed at the oriDa origin in Gal4 and Gal4–Orc5 cell lines as detected by amplification with the D region primers. (B) H4K12 acetylation observed at the UAS in Gal4 and Gal4–Orc5 cell lines as detected by amplification with the qUAS4 region. (C) Agarose gel showing an example of the ChIP analysis for the UAS and control (alpha) regions. Antibodies against V5 (as a positive control) or acetylated H4K12 (AcH4) were used as indicated. The left-hand side of the gel shows results for the Gal4 cell line and the right-hand side for the Gal4–Orc5 cell line. In both cases the first three lanes show titration of the input DNA.

Article Snippet: A rabbit polyclonal antibody against histone H4K12 was purchased from Abcam (Ab1761).

Techniques: Negative Control, Amplification, Agarose Gel Electrophoresis, Control, Positive Control, Titration

Journal: Nature protocols

Article Title: Creating localized DNA double-strand breaks with microirradiation.

doi: 10.1038/nprot.2010.183

Figure Lengend Snippet: Figure 2 | Cells covered by a micropore membrane. Cells cultured on 22 × 22-mm cover slips (left) are covered by a micropore membrane (right). It is crucial to aspirate as much PBS as possible (see Fig. 1b). Care should also be taken not to trap bubbles between the membrane and cells. Some cells may be left uncovered so that BrdU labeling and UVC dose can be checked by the pan-nuclear signal of phosphorylated histone H2AX (Fig. 1c).

Article Snippet: BrdU (Boehringer Mannheim, cat. no. 280879) Mouse antiphosphorylated histone H2AX at serine 139 monoclonal antibody (BioLegend, clone 2F3, cat. no. 613402) Mouse antiphosphorylated ATM at serine 1981 monoclonal antibody • • • • © 20 11 N at u re A m er ic a, In c. A ll ri g h ts r es er ve d . 136 | VOL.6 NO.2 | 2011 | nature protocols (Rockland, clone 10H11.E12, code no. 200-301-400) Rabbit antiphosphorylated histone H2AX at serine 139 antibody (Bethyl, cat. no. A300-081A) Rabbit anti-p53 binding protein 1 (53BP1) antibody (Bethyl, cat. no. A300-272A) Rabbit anti-ring finger protein 8 (RNF8) polyclonal antibody (Abcam, cat. no. ab4183) Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, cat. no. A11001) Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen, cat. no. A11008) Tris (Wako, cat. no. 207-06275) NaCl (Wako, cat. no. 191-01665) Tween-20 (Wako, cat. no. 167-11515) Skim milk (BD Difco, cat. no. 232100) Formalin (Wako, cat. no. 064-00406) ! cautIon Toxic by inhalation; work in a fume hood.

Techniques: Membrane, Cell Culture, Labeling

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Olaparib and ionizing radiation trigger a cooperative DNA-damage repair response that is impaired by depletion of the VRK1 chromatin kinase

doi: 10.1186/s13046-019-1204-1

Figure Lengend Snippet: List of antibodies and conditions used in this work

Article Snippet: Histone H4-K16 ac , Rabbit monoclonal , 1/500; 1/400 , ab109463 , Abcam.

Techniques:

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule approach for the enrichment of plasma-circulating H3-K27M-mutant nucleosomes (A) Experimental scheme: nucleosomes are extracted from HEK293 cells ectopically expressing either WT or mutant H3, and the DNA is fluorescently labeled with Cy3 by Klenow polymerase and Cy3-dATP. Next, labeled H3-K27M nucleosomes are captured on PEG-streptavidin surface covered with biotinylated antibodies targeting the H3-K27M mutant histone (α-H3-K27M) and imaged by TIRF imaging. Nucleosomes containing WT H3 do not bind the microscope coverslip and are washed prior to imaging. (B) Representative TIRF images of Cy3 signal, corresponding to surface-bound nucleosomes, extracted from HEK293 cells expressing WT or mutant histone. Nucleosomes extracted from cells expressing H3-K27M were loaded also on a surface absent of α-H3-K27M antibodies (middle), to demonstrate antibody-dependent capture. Numbers within images represent counted spots; each spot corresponds to a single surface-bound nucleosome. Scale bar applies to all three images. Nucleosomes extracted from cells expressing mutant H3 show higher binding to surface coated with α-H3-K27M antibodies. (C) Quantification of the experiment described in (B). Data are presented as the mean ± SD of 50 fields of view (FOVs) per sample. (D) Experimental scheme: cfNuc from plasma are immobilized on a PEG-streptavidin covered with biotinylated α-H3-K27M antibody. Captured H3-K27M nucleosomes are detected by incubation with fluorescently labeled antibody followed by TIRF imaging. (E) Representative TIRF images of different antibodies targeting the indicated histone PTMs, while incubated on surfaces enriched for plasma-circulating H3-K27M nucleosomes (as described in D). Antibodies were tested on plasma samples from healthy individuals, as well as patients with DMG carrying WT H3 or H3-K27M. Numbers within images represent counted spots; each spot corresponds to a single surface-bound nucleosome. Scale bar applies to all images. H3K27ac and H3K36me2 antibodies are suitable for the detection of surface-bound H3-K27M nucleosomes. (F and G) Quantification of the single-molecule signal obtained for H3K27Ac antibody when incubated with and without H3-K27M enriched cfNuc from different plasma samples. Data are presented as the mean ± SD of 50 FOVs per sample.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Expressing, Labeling, Imaging, Microscopy, Binding Assay, Incubation

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule detection of mutant nucleosomes in plasma differentiates patients with H3-K27M from those with H3 WT DMG (A) Plasma samples of patients with DMG carrying WT H3 or H3-K27M mutant (light blue and dark blue, respectively) and plasma of healthy adults or children (light gray and dark gray, respectively) were analyzed as described in Figure 2 D. Mutant nucleosomes captured on surface were detected by H3K27ac antibody. Each bar represents a subject, and data are presented as the mean of the normalized H3K27ac counts ±SD of 50 FOVs per sample. (B) Boxplot representation of the data in (A), with samples grouped as follows: DMG H3-K27M ( n = 52), H3 WT glioma ( n = 16), healthy adults ( n = 21), and healthy children ( n = 29). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.5× the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test and adjusted by Bonferroni correction for multiple comparisons. ∗∗ p value <0.01, ∗∗∗ p value <0.001; ns, non-significant. (C) Receiver operating characteristic (ROC) curve discriminates between plasma samples containing WT H3 nucleosomes ( n = 65) and H3-K27M-mutant nucleosomes ( n = 52) using a naive Bayes model. The area under the curve (AUC) for H3-K27M-H3K27ac normalized counts (blue line), while the red dashed diagonal line indicates expected curve for random classification. Blue shaded area represents 95% confidence interval. (D) H3-K27M-H3K27ac classifier performance; each bar represents the mean value of repeated ( n = 10,000) 4-fold cross-validation across all samples.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Detection of plasma-circulating mutant p53 in patients with DMG (A) Experimental scheme: biotinylated capture antibodies targeting total p53 protein (the WT and mutant form) are anchored to a PEG-streptavidin surface (α-p53). Plasma-circulating p53 protein molecules are captured on surface, followed by incubation with two distinct fluorescently labeled p53 antibodies: an antibody targeting all forms of p53 (“total p53,” red) and an antibody specific to the mutant conformation of p53 (“mutant p53,” green). (B) Representative TIRF images of the indicated p53 antibodies incubated on surfaces enriched for plasma-circulating p53 proteins. Scale bar applies to all images. (C) A cohort of 19 plasma samples of H3-K27M DMG patients was analyzed as described in (A) (13 samples harbor TP53 mutations and 6 harbor WT TP53). Principal component analysis (PCA) with the following input parameters: normalized counts of total p53 and mutant p53, and the ratio between mutant and total p53. Sample groups are color-coded according to known TP53 status; each dot represents one plasma sample. (D) The ratio between mutant and total p53 signal for each sample is shown. Each bar represents a sample, color-coded according to known TP53 status. Data are presented as the mean ± SD of 50 FOVs per sample. (E) Boxplot representation of the data in (D) grouped according to TP53 status (mutant p53 n = 13, WT-p53 n = 6). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.5× the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test. ∗∗∗ p value <0.001.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Incubation, Labeling

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule measurements of mutant nucleosomes as a potential proxy for tumor growth (A) Experimental scheme for parallel measurements of cell viability and H3-K27M-mutant nucleosomes released to culture media. Cells were plated as monolayer, and a day later media was replaced to initiate the experiment. Media was collected for single-molecule measurements and replaced to account for newly released nucleosomes between examined time points. Viability measurements were conducted from a parallel culture. (B) Viability measurements and H3-K27M-H3K27ac single-molecule measurements are shown for untreated culture (left) and 5 μM ONC201-treated culture (right). The drug was replenished every 2 days. For single-molecule measurements, mean ± SD of 40 FOVs for each time point is shown. For CellTiter-Glo measurements, mean ± SE of three technical repeats for each time point is shown. (C) Clinical features, initial MRI, and MRI of disease at clinical progression of DMG patient UMPED 128. (D–J) Analysis of serial plasma samples from patients undergoing ONC201/206 treatment. Single-molecule measurements (normalized to first data point) of mutant nucleosomes (H3-K27M-H3K27ac) were performed on serial time point plasma samples plotted along with tumor cross-sectional area according to MRI. Green bars: ONC201/206 mainline treatment. Purple bars: radiation treatment. Brown bars: other treatment modalities that are detailed in Table S1 . Radiographic progression is marked with a black asterisk placed next to the corresponding MRI data point. Day 0 corresponds to the initiation of treatment. (K) Heatmap showing the correlation between the direction of change for the indicated features (see ). For the majority of patients, tumor MRI measurements show the highest correlation with the single-molecule measurements of H3-K27M-H3K27ac in plasma. The correlation was calculated based on interpolated data points to account for measurements not taken at exactly the same time. See also mutant p53 and ctDNA data in Figure S4 .

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet:

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Recombinant, Conjugation Assay, Antibody Labeling, Software