Journal: Cancers

Article Title: Advances in Prognostic Methylation Biomarkers for Prostate Cancer

doi: 10.3390/cancers12102993

Figure Lengend Snippet: Methylation profiling approaches for biomarker discovery and validation. ( A ) Candidate gene approaches: Targeted candidate gene approaches used in a priori studies and for the validation of novel candidate markers. ( B ) Genome wide approaches: Microarray and sequencing-based genome-wide approaches used for the discovery of novel biomarkers—comparison of the methodology, number of CpGs (cytosine-guanine dinucleotides) and/or genes targeted and coverage of the methylome across the different platforms. RefSeq: Reference Sequences; PCR: polymerase chain reaction; MSP: methylation-specific PCR; ddPCR: droplet digital PCR; COBRA: combined bisulphite restriction analysis; qMSP: quantitative methylation-specific PCR; HM: human methylation; RRBS: reduced representation bisulphite sequencing; MBDCap-Seq: methyl-CpG binding domain capture sequencing; WGBS: whole genome bisulphite sequencing.

Article Snippet: These studies used targeted methylation profiling techniques including methylation-specific PCR (MSP) [ ], quantitative methylation-specific PCR (qMSP) [ ], pyrosequencing [ , ] and mass spectrometry (MassARRAY EpiTYPER, Agena Bioscience, San Diego, California, USA) [ ] to assess the methylation profile of a specific gene of interest ( ). lists other targeted approaches that have also been used to assess methylation in cancer, including ddPCR [ ], COBRA [ ], high resolution melt curve [ ] and headloop MSP [ , , ].

Techniques: Methylation, Biomarker Discovery, Genome Wide, Microarray, Sequencing, Comparison, Polymerase Chain Reaction, Digital PCR, Combined Bisulfite Restriction Analysis Assay, Bisulfite Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Diagram of growth competition assay. mCherry-labelled RPE1 cells were mixed with unlabeled TP53 −/− RPE1 (1:1), exposed to IR and grown for 6 days. b, Relative abundance of unlabeled TP53 −/− Clone#1 measured by Intellicyte high-throughput cytometry ± SEM (n=6) is shown, normalized to the untreated (0Gy) cohort at each time point. c, Relative abundance of unlabeled TP53 −/− Clone#2 ± SEM (n=6) is shown, normalized to the untreated (0Gy) cohort at each time point. d, Representative immunofluorescence images of 53BP1 foci in cells with indicated genotypes untreated (no IR) or treated with IR (5Gy) and collected at .5, 2, and 4 h after irradiation. e, Quantification of 53BP1 foci. Data shown are mean (n=50 cells per treatment condition) ± SEM (n=3), and are consistent across two independent biological replicates. * p <0.05; ** p <0.01; by two-tailed Student’s t-test. f, Representative Neutral COMET fluorescence staining for DNA tails in cells with indicated genotypes treated with or without 5Gy IR. For irradiated cells, 2 timepoints are shown: immediately after and 4 hours post IR. COMET tails and heads are denoted by OpenComet software analysis. g, Quantification of DNA DSBs via Neutral COMET assay reported as tail DNA percent at 0 and 4 hours post IR in RPE1 and two TP53 −/− RPE1 cell lines. Data shown are mean (n= 50-150 cells per treatment condition) ± SEM, and are consistent across three independent biological replicates. * p <0.05; ** p <0.01; **** p <0.0001 by two-tailed Student’s t-test.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Competitive Binding Assay, High Throughput Screening Assay, Cytometry, Immunofluorescence, Irradiation, Two Tailed Test, Fluorescence, Staining, Software, Neutral Comet Assay

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Live cell imaging procedure. Cells transfected with 10 nM si-control or si- TP53 for 48 h prior to imaging. 18 h into imaging, cells are treated with NCS (100 nM), DNA-PKi (.5 uM) or both and imaged for 72 total hours. b, RPE1 cell expressing the PCNA-mCherry and 53BP1-mVenus reporters. Cell cycle phases delineated by PCNA foci and DNA DSBs are marked by 53BP1 foci. c, RT-qPCR for TP53 mRNA levels (left) and CDKN1A mRNA levels (right) in si-control treated vs. si- TP53 treated cells. To induce CDKN1A expression, cells irradiated at 5Gy and mRNA harvested 3 hrs post IR. d, Heatmap of 53BP1 foci tracings for single cells tracked from birth to mitosis or end of imaging. For si-control (n = 30 cells) and si- TP53 treated RPE1 (n = 60 cells) treated with NCS 100 ng/ml. For visualization, cells are aligned to 10 frames prior to drug addition (black arrow). e, Heatmap of 53BP1 foci tracings for si-control (n = 25 cells) and si- TP53 treated RPE1 cells (n = 55 cells) treated with 100 ng/ml NCS + 0.5 uM DNA-PKi. f, Peak 53BP1 foci counts for cells treated with 100 ng/ml NCS or NCS+0.5 uM DNA-PKi. Significance determined using two-tailed t-test. g, Area under the curve (AUC) analysis of 53BP1 burden showing integral DNA damage for cells treated with NCS vs. NCS and DNA-PKi. Cells are segregated into two groups: cells exposed to drug in G1 vs. S phase (n = 25-30 G1or S cells for si- TP53 cohort, n = 10-15 G1 or S cells for si-control cohort). Significance determined by two-tailed t-test. **** p <0.0001, *** p <0.001, n.s. = non-significant. h, 53BP1 foci burden in G1 vs. S phase p53-deficient RPE1 upon exposure to NCS and DNA-PKi. Dashed line = S phase onset, blue line = mean 53BP1 foci burden for all cells in G1 with NCS and DNA-PKi addition, orange line = mean foci value for cells in G1 with NCS treatment alone, (n = 30 cells for each condition).

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Live Cell Imaging, Transfection, Control, Imaging, Expressing, Quantitative RT-PCR, Irradiation, Two Tailed Test

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Normal Mitosis: RPE1 Cell cycle representative of normal mitosis, with NCS treatment only. For all cells in this figure both the PCNA and the 53BP1 channels are shown as two individual movies. b, Transient G2 Delay: RPE1 cell cycle representative of a transient cell cycle delay in G2 (length of G2 is significantly prolonged in comparison to untreated cells). This cell was treated with NCS and DNA-PKi. c, G1 Arrest: RPE1 cell cycle representative of a permanent G1 arrest. This is a p53 proficient cell treated with NCS and DNA-PKi.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Comparison

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Cell cycle outcome analyses for si-control treated RPE1, dashed white line indicates drug addition, each row is an individual cell (n = 60 cells for NCS and n=60 cells for NCS+DNA-PKi treatment). Colored bars indicate different phases of the cell cycle, legend shown with no treatment control for comparison. Cells with red bars at the end of mitosis indicate terminal cell cycle event (mitotic catastrophe or apoptosis). Event frequency is reported as a percentage on the right. Cells exposed in G1 vs. S cells are treated as separate cohorts. Fisher’s exact test was performed between −/+ DNA-PKi cohorts using 2 outcome groups (viable, vs. non-viable (arrested cells + terminal outcomes). **** p <0.0001, n.s. =non-significant b, Cell cycle outcome analyses for si- TP53 treated RPE1, dashed line indicates drug addition, each row is an individual cell (n = 60 cells for NCS and n=60 cells for NCS+DNA-PKi treatment). c, AUC analysis of 53BP1 damage burden in viable vs. non-viable p53-deficient cells that were treated with NCS and DNA-PKi. Statistical significance was calculated using a Mann-Whitney test comparing ranks. **** p <0.0001 d, Dynamics of 53BP1 foci burden p53-deficient RPE1 segregated by mitotic viability. The red line corresponds to mean 53BP1 foci burden for all p53-deficient cells treated with NCS and DNA-PKi that undergo catastrophic mitoses, black line indicates mean foci value for p53-deficient cells with NCS and DNA-PKi treatment that are viable post mitosis, (n = 20 viable cells and n = 33 non-viable cells).

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Time stamped image sequence of apoptotic cell (PCNA channel shown). Cells that experienced nuclear degradation during cell cycle prior to mitosis were categorized as “apoptotic cells.” In this sequence a cell in G2 experiences cell death at 27 hours post birth, with indication of mitotic attempt, with nuclear envelope collapse or presence of any daughter cells. b, Time stamped image sequence of cell that experienced mitotic catastrophe (PCNA channel shown). Cell undergoes nuclear envelope collapse (24:10), and attempts mitosis, in subsequent images fragmentation of nucleus is clearly visible with no viable daughter cells present. Cell non-viability during mitosis was defined as mitotic catastrophe. c, Integral DNA damage burden for p53-deficient cells treated with NCS (100 ng/ml) and DNA-PKi (.5 uM) are calculated and segregated by viable (black) vs. non-viable outcomes (red). Legend indicates which phase of cell cycle the cells are in during drug exposure, followed by the phase for which the burden is calculated. Ex: G1 cells G1 burden = cells in G1 during drug exposure and total damage burden in G1. Area under the curve (AUC) analysis was performed by plotting 53BP1 foci counts over time for each cell and integrating burden over time. Statistical significance was determined using two-tailed Student’s t-test.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Sequencing, Two Tailed Test