Journal: Scientific Reports

Article Title: Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461

doi: 10.1038/s41598-021-88988-w

Figure Lengend Snippet: Pooled CRISPR-Cas9 screens reveal genetic determinants of sensitivity to G4 drugs. ( a ) Schematic of pooled screen. ( b ) Experimental design of drug screens. HCT116 cells were transfected with the LCV2-puro library at an MOI of 0.3. After 24 h, 0.5 µg/ml puromycin was added and the cells kept in puromycin for 3 days. On day 5 drug/vehicle treatments were started in triplicate and continued until day 19. Cells were passaged with freshly added vehicle or drugs, and harvested on days 1, 4, 5, 12, 15 and 19 to make DNA for the downstream sequencing library preparation. ( c ) Top 81 depleted genes from MAGeCK output. Each row represents the corresponding drug treatment groups. Heatmap shows the MAGeCK depletion scores of the genes. The heatmap is ordered according to the depletion scores of CX-5461 (IC 50 ) group, ranking the most depleted genes on the left. To emphasize genes with the highest depletion score, the median of the color gradient (white) was shifted to 0.2. ( d ) UpSet plot showing the intersection of candidate genes undergoing negative selection with G4 stabilizers (MAGeCK P -values < 0.045) within each drug condition. In the bottom matrix, each row represents the set of genes identified for the indicated drug condition, and each column represents the intersection of the drug condition gene sets indicated by black dots. The histogram to the right of the matrix indicates the number of genes identified for each input drug condition. The histogram on the top shows the number of genes in each intersection set. Gene hits common in all CX-5461 and PDS conditions are shown in yellow.

Article Snippet: Individual sgRNAs were designed using the Deskgen Cloud CRISPR Design Software (Desktop Genetics). sgRNA cloning into LCV2-DRX2 vector was performed as previously described , .

Techniques: CRISPR, Transfection, Sequencing, Selection

Journal: Cancer research

Article Title: Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

doi: 10.1158/0008-5472.CAN-19-1036

Figure Lengend Snippet: A) iPOND analysis of newly replicated DNA association with mtp53 R273H and PARP in MDA-MB-468.vector, MDA-MB-468.shp53 cells and mtp53R273H overexpression MDA-MB-468.shp53+R273H cells (left panel) and PANC-1 cells (right panel). 1 × 108 cells were labeled with 10 μM EdU for 45 min. The protein-DNA complexes were cross-linked, nascent DNA was conjugated to biotin using click chemistry, and protein-DNA complexes were purified. The eluted proteins were analyzed using western blot. A sample that did not include biotin-azide was used as a negative control. Representative of two independent experiments. B) Single cell in situ proximity ligation assay (PLA) with pulse EdU labeling showed mtp53 R273H associated with newly replicated DNA throughout S-phase. Cells were labeled with 125 μM EdU for 15 min. The protein-DNA complexes were cross-linked, nascent DNA was conjugated to biotin using click chemistry. Representative maximum intensity projection images of 2 central slices for S-phase progression of MDA-MB-468 (upper left panel) and PANC-1 (upper right panel) are shown; thickness: 2μm. PLA of mtp53 R273H/EdU (red) was performed using anti-biotin and anti-p53 antibodies. Anti-biotin IF (green) indicates cells undergoing DNA synthesis. DNA was counterstained with DAPI (blue). Analysis of mtp53/EdU foci per nuclei by CellProfiler and S-phase progression for each nucleus was manually grouped into Early, Mid, or Late S-phase using GraphPad Prism 8. Statistical analysis for MDA-MB-468 and PANC-1: * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01, **** represents a p-value ≤ 0.0001, ns= not significant. The p-value was determined by 2-tailed student t-test. MDA-MB-468: Early: n=86, Mid: n=49, Late: n=69; PANC-1: Early: n=51, Mid: n=23, Late: n=44. Representative of three independent experiments with two technical replicates each. C) The z-stack maximum intensity projection images showed mtp53 R273H and PARP associated with newly replicated DNA in MDA-MB-468 cells. Cells were labeled with 125 μM EdU for 15 min with or without 100 μM thymidine chases for 60 min. PLA with EdU (red) was performed as in Fig. 1B. Three independent experiments were performed. D) PLA with or without 20 min incubation of 10 μM poly(ADP-ribose) glycohydrolase inhibitor (PARGi) and 10 μM EdU labeling. Maximum intensity projection images are shown. Representative of two technical replicates.

Article Snippet: Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC beads to precleared lysates and incubated at 4°C for 2 hr with rotation.

Techniques: Plasmid Preparation, Over Expression, Labeling, Purification, Western Blot, Negative Control, In Situ, Proximity Ligation Assay, DNA Synthesis, Incubation

Journal: Cancer research

Article Title: Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

doi: 10.1158/0008-5472.CAN-19-1036

Figure Lengend Snippet: A) Analysis of p53/PARP1 complexes (red) by immunofluorescence microscopy in combination with in situ proximity ligation assay (PLA) in MDA-MB-468 and MCF7 cells. DNA was counterstained with DAPI (blue). The z-stack maximum intensity projection images are shown. Two independent experiments were performed. B) Analysis of fluorescent foci per cell by CellProfiler software and demonstrated by scatter plot using Prism7 GraphPad. **** represents p-value ≤ 0.0001. The p-value was determined by 2-tailed Student t-test. Two independent experiments were performed. C) p53 protein level in MCF7 and MDA-MB-468 cells. Two independent experiments were performed. D) Co-Immunoprecipitation (Co-IP) of PARP and MCM7 with mtp53 in MDA-MB-468 cells. Two independent experiments were performed. E) MTT assay showed reduction of mitochondrial activity after combination treatment 1mM temozolomide plus 10 μM talazoparib for 24hr in cells with mtp53 R273H overexpression compared to cells with endogenous mtp53. Cells were seeded at 1.25 × 105 cells per well in 12-well plates and attached overnight. Cells were treated with either 10 μM talazoparib or 1mM temozolomide, or both for 24 hrs. The absorbance was quantified by measuring the absorbance at 550 nm subtracted from the absorbance at 620 nm. All MTT data are represented as mitochondrial dehydrogenase activity as percentage of a dimethyl sulfoxide (DMSO) vehicle-treated control. Three independent experiments were performed with two technical replicates. F) Chromatin protein levels of mtp53, 53BP1 and Fibrillarin were determined by Western blot analysis with or without combination treatment Temo plus Tal in MDA-MB-468 mtp53 R273H overexpression compared to cells with endogenous mtp53. 50 μg of chromatin protein were loaded on 10% SDS-PAGE gel. Two independent experiments were performed.

Article Snippet: Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC beads to precleared lysates and incubated at 4°C for 2 hr with rotation.

Techniques: Immunofluorescence, Microscopy, In Situ, Proximity Ligation Assay, Software, Immunoprecipitation, Co-Immunoprecipitation Assay, MTT Assay, Activity Assay, Over Expression, Control, Western Blot, SDS Page

Journal: Cancer research

Article Title: Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

doi: 10.1158/0008-5472.CAN-19-1036

Figure Lengend Snippet: A) Analysis of protein expression correlation of p53 and PARP1 in breast invasive carcinoma samples from The Cancer Genome Atlas (TCGA) database40. A total of 817 breast tumor samples were profiled and 633 cases were also profiled by reverse-phase protein array (RPPA). ER, PR and HER2 status was clinically determined by immunohistochemistry. Scatter plots showed p53 and PARP1 protein expression correlation analysis by protein level (z-score) determined by RPPA. Red: TNBC; Green: estrogen receptor–positive (ER+) patients. B) Left panel: Examples of manually scored p53 and PARP1 staining intensity categories: no staining (0), weak staining (1), moderate staining (2) and strong staining (3) at magnification 200x. Right top panel: dot plot diagram showed immunohistochemical (IHC) staining of p53 score in tissue microarray (TMA) from Basal-like, Luminal A and Luminal B subtype of breast cancer. Right lower panel: Dot plot diagram showed immunohistochemical (IHC) staining of PARP1 score in tissue microarray (TMA) from Basel-like and Luminal A subtype of breast cancer grouped by p53 level. C) Live cell imaging of MDA-468 and MCF7 cells PARPi-FL (Green). Cells were imaged by confocal microscopy after 20 min incubation of 500 nM of PARPi-FL. Hoechst staining (blue) used to visualize nuclei. Two independent experiments were performed. D) Protein levels of p53, PARP1 and PAR in breast cancer PDX model WHIM6 and WHIM25 were determined by western blot analysis. Whole cell proteins were extracted from NSG mice xenograft tumors and 50ug of protein was loaded on 10% SDS-PAGE gel. Three technical replicates were performed.

Article Snippet: Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC beads to precleared lysates and incubated at 4°C for 2 hr with rotation.

Techniques: Expressing, Protein Array, Immunohistochemistry, Staining, Immunohistochemical staining, Microarray, Live Cell Imaging, Confocal Microscopy, Incubation, Western Blot, SDS Page

Journal: Molecular Systems Biology

Article Title: Essentiality and dynamic expression of the human tRNA pool during viral infection

doi: 10.1038/s44320-025-00181-7

Figure Lengend Snippet: ( A ) A pie chart describing the different sgRNA sub-libraries. For each sub-library, the number of targeted genes and sgRNAs is specified (the number of genes is listed in brackets, and the number of sgRNAs is indicated within the pie chart). As non-targeting sgRNAs do not correspond to existing genes, the number of genes for this sub-library is not mentioned. ( B ) A volcano plot showing targeted gene hits from tRNA-CRISPR screen of HFF cell growth. The x-axis shows the Z-score of log2 fold change (FC) between competing cells (3 days of competition) and ancestor samples (median of log2 FC for all high-ranked sgRNAs per gene). The y-axis shows the –log10 FDR as calculated using the MAGeCK tool. The genes are marked according to the sub-libraries. Significance is determined by FDR < 0.05 and marked with dashed gray lines. All values are calculated for three biological repeats. ( C ) A heat map showing the (−log10) p -value of the hypergeometric test, which tests the enrichment of each sub-library in one of the following groups: significantly depleted in competing cells (log2FC < 0, FDR < 0.05) or significantly enriched in competing cells (log2FC > 0, FDR < 0.05). ( D ) Differences in the essentiality of tRNA isodecoders that are the sole targets of their corresponding sgRNA. tRNA essentiality is determined by the median log2 FC of its corresponding sgRNA between competing and ancestor cells. The arrow points towards higher tRNA essentiality. tRNA isoaceptors are denoted on the x-axis. Each dot corresponds to a different isodecoder type within the isoaceptor family. The number of isodecoders per tRNA family is mentioned in parentheses. ( E ) Comparison between the (log2) expression of the tRNA isodecoder in HFF (x-axis) and their essentiality, as shown in Fig. 5D (y-axis). The arrow points towards higher tRNA essentiality. Pearson correlation r = −0.41, p -value = 0.04.

Article Snippet: The CRISPR sgRNA design tool in Benchling ([Biology Software]. (2022), retrieved from https://benchling.com ) was used for sgRNA design, utilizing the default parameters.

Techniques: CRISPR, Comparison, Expressing

Journal: Molecular Systems Biology

Article Title: Essentiality and dynamic expression of the human tRNA pool during viral infection

doi: 10.1038/s44320-025-00181-7

Figure Lengend Snippet: ( A ) A schematic representation of the experimental setup and distributions of the measured GFP levels of uninfected HFF cells (upper panel), HCMV-infected HFF cells with non-targeting sgRNA (middle panel), and CRISPR-targeted and HCMV-infected HFF cells (lower panel). The percentage of cells in each of the GFP levels, GFP1-4, is marked in the middle and lower panels. ( B ) A volcano plot for targeted gene hits from tRNA-CRISPR screen in HCMV infection. The x-axis shows the Z-score of log2 FC between lowly infected cells (GFP2) and highly infected cells (GFP4). The y-axis shows the –log10 FDR as calculated from MAGeCK. The genes are marked according to the sub-libraries. Significance is determined by FDR < 0.05. All values are calculated for three biological repeats. ( C ) A heat map showing the (−log10) p -value of the hypergeometric test, which tests the enrichment of each sub-library in one of the following groups: significantly enriched in GFP2 cells (log2FC > 0, FDR < 0.05) or significantly depleted in GFP2 cells (log2FC < 0, FDR < 0.05). ( D ) The number of viral genomes estimated by the relative number of UL44 normalized to the B2M human gene, calculated by qPCR, in each individual CRISPR knockout. The dots in each bar depict three or four biological repeats. P -values represent statistical significance of differences between each group and the non-targeting control, as evaluated using Welch’s t-test with Holm correction for multiple comparisons. ( E , F ) Expression level (RPM) determined from tRNA-sequencing of ( E ) iMet-CAT-1-1 gene and ( F ) His-GTG-1-1 gene in cells targeted by non-targeting sgRNA (NT) or by sgRNAs corresponding to the tested tRNA. The dots in each bar depict three biological repeats. ( G ) A heat map describes the z-score log2FC between the different lowly infected cell populations (GFP1, GFP2, GFP3) and the highly infected cells (GFP4) for the significant gene hits. Genes found as significant hits in at least one of the comparisons are presented here. Non-significant hits are marked in gray squares. The genes are colored and marked according to their sub-library, corresponding to the colors and marker shapes described in the legend of Fig. 6B. The dendrogram depicts the similarity between the comparisons based on the enrichment gene hits pattern.

Article Snippet: The CRISPR sgRNA design tool in Benchling ([Biology Software]. (2022), retrieved from https://benchling.com ) was used for sgRNA design, utilizing the default parameters.

Techniques: Infection, CRISPR, Knock-Out, Control, Expressing, Sequencing, Marker