Journal: Cancers

Article Title: Optimization of New Catalytic Topoisomerase II Inhibitors as an Anti-Cancer Therapy

doi: 10.3390/cancers13153675

Figure Lengend Snippet: T637 and T638 are TOP2A inhibitors. ( A ) K-DNA decatenation assays and ( B ) supercoiled plasmid DNA relaxation assays are used to measure TOP2A enzyme activity in the presence of 0.01–200 uM T637 or T638. Three biological repeats were performed with each concentration of the compounds, and the densitometry of the D-DNA bands from ( A ) and the SC bands from ( B ) was used to establish an inhibition curve for IC 50 calculation.

Article Snippet: TOP2A(431-1193) protein samples were labeled with the red fluorescent dye NT647 using the Monolith NT Protein labeling kit RED-NHS amine-reactive (NanoTemper Technologies, München, Germany).

Techniques: Plasmid Preparation, Activity Assay, Concentration Assay, Inhibition

Journal: Cancers

Article Title: Optimization of New Catalytic Topoisomerase II Inhibitors as an Anti-Cancer Therapy

doi: 10.3390/cancers13153675

Figure Lengend Snippet: T638 does not intercalate with DNA but binds to TOP2 protein. ( A ) Ethidium bromide displacement assays were performed by mixing 100 µg/mL salmon sperm DNA with −/+2 µg/mL ethidium bromide and the indicated compounds. Reactions were subjected to fluorescence emission of 590 nm and excitation of 535 nm. Obtained signals were detected by using the Multimode Microplate Reader (Infinite F500). ( B ) MST assay was performed by incubating 5 nM of the purified human TOP2A(431-1193) labeled with the red fluorescent dye NT647 in the presence of vehicle or 0.015–500 µM of T638. Reactions were subjected to 5–20% LED/excitation power and medium MST power using premium capillaries. Obtained signals were detected by using the Monolith NT.115 system. The change in thermophoresis is expressed as the change in the normalized fluorescence (Δ F norm ). MST assays were repeated with two control compounds, enzalutamide (Enza) and merbarone.

Article Snippet: TOP2A(431-1193) protein samples were labeled with the red fluorescent dye NT647 using the Monolith NT Protein labeling kit RED-NHS amine-reactive (NanoTemper Technologies, München, Germany).

Techniques: Fluorescence, Purification, Labeling

Journal: Cancers

Article Title: Optimization of New Catalytic Topoisomerase II Inhibitors as an Anti-Cancer Therapy

doi: 10.3390/cancers13153675

Figure Lengend Snippet: T637 and T638 block TOP2–DNA interaction and prevent TOP2 mediated DNA cleavage. ( A ) EMSA was performed by purified full-length TOP2A or TOP2B proteins at a contraction of 300 nM with IRDay700 labeled 5ZRF DNA oligo at a concentration of 100 nM in the presence of −/+200 µM of ICRF193, T60, T637, and T638. ( B ) EMSA was performed by incubating 2 uM of purified human TOP2A(431-1193) with 300 nM of IRDay700 labeled 5ZRF DNA oligo in the presence of −/+T60, T637, T638, or merbarone. ( C ) FP assay was performed by incubating 500 nM of the purified human TOP2A(431-1193) with 1 nM of 3’ 6-FAM labeled 5ZRF DNA oligo in the presence of 0.01–200 µM of T60, T637, T638, or merbarone. Reactions were subjected to fluorescence emission of 485 nm and excitation of 535 nm, which were detected by using the Multimode Microplate Reader (Infinite F500). ( D ) DNA cleavage assays were performed using full-length human TOP2A and TOP2B incubated with the supercoiled pHOT plasmid in the presence of 200 µM of etoposide plus 1, 100, or 200 µM of ICRF193, T60, T637, and T638. Reactions were separated on a DNA gel with ethidium bromide. All experiments were repeated three times, and one representative image was shown. Note: N, nicked DNA; L, linear DNA; and SC, supercoiled DNA. Full Website Blot Figures are shown in the .

Article Snippet: TOP2A(431-1193) protein samples were labeled with the red fluorescent dye NT647 using the Monolith NT Protein labeling kit RED-NHS amine-reactive (NanoTemper Technologies, München, Germany).

Techniques: Blocking Assay, Purification, Labeling, Concentration Assay, FP Assay, Fluorescence, Incubation, Plasmid Preparation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Proteomic Analysis Reveals That Snail Regulates the Expression of the Nuclear Orphan Receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and Interleukin 17 (IL-17) to Inhibit Adipocyte Differentiation *

doi: 10.1074/mcp.M114.045328

Figure Lengend Snippet: Function analysis of selected deregulated proteins in Snail-transfected cells

Article Snippet: In total, 458 out of 2251 quantified proteins in 3T3-L1 were previously observed in nucleus. . Network and Pathways Alterations Induced by Snail Among other functions, Snail-deregulated proteins were involved in chromatin remodeling such as Hmga2, Top2A (up-regulated), or chromobox proteins such as CBX1, 3, 5 (up-regulated), 6 and 8 (down-regulated) ( A ) ( ) ( supplemental Table S2 ).

Techniques: Multiplex sample analysis

Journal: Breast Cancer Research : BCR

Article Title: Evaluating the predictive value of biomarkers for efficacy outcomes in response to pertuzumab- and trastuzumab-based therapy: an exploratory analysis of the TRYPHAENA study

doi: 10.1186/bcr3690

Figure Lengend Snippet: The relationship between biomarker levels and whether a pathologic complete response (pCR) was achieved, adjusted for estrogen receptor status (all arms pooled)

Article Snippet: Targos Molecular Pathology GmbH, Kassel, Germany conducted analysis of: mRNA expression levels of HER2 , HER3, EGFR, amphiregulin, and betacellulin ; protein expression levels of HER3, EGFR, IGF-1R, and PTEN and of c-myc and TOP2A gene amplification.

Techniques: Biomarker Discovery, Membrane, Mutagenesis

Journal: Breast Cancer Research : BCR

Article Title: Evaluating the predictive value of biomarkers for efficacy outcomes in response to pertuzumab- and trastuzumab-based therapy: an exploratory analysis of the TRYPHAENA study

doi: 10.1186/bcr3690

Figure Lengend Snippet: Comparison of baseline levels of biomarkers derived from tissue samples with the levels detected at surgery. (A) PTEN nuc, (B) HER2-CR, ( C ) EGFR-CR, (D) HER2-mem. (In the box plot, the horizontal line represents the median value, the diamond represents the mean, the upper and lower bounds of the box represent the 75th and 25th quartiles, respectively, and whiskers represent 95% confidence limits). CR, concentration ratio; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; mem, membrane; nuc, nuclear; PTEN, phosphatase and tensin homolog.

Article Snippet: Targos Molecular Pathology GmbH, Kassel, Germany conducted analysis of: mRNA expression levels of HER2 , HER3, EGFR, amphiregulin, and betacellulin ; protein expression levels of HER3, EGFR, IGF-1R, and PTEN and of c-myc and TOP2A gene amplification.

Techniques: Comparison, Derivative Assay, Concentration Assay, Membrane

Journal: Nature Communications

Article Title: Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

doi: 10.1038/s41467-024-53877-z

Figure Lengend Snippet: A IMPDH2 shown in red, scored as an interactor of PARP1 and TOP2A DNA damage effectors (from OpenCell resource). Purine pathway enzymes are shown in orange, rest of metabolic enzymes in yellow. B Western blot of the chromatin fraction from MDA-MB-231 cells of IMPDH2 and IgG immunoprecipitation showing PARP1 and TOP2A pull-down after 3 h of 1 μM etoposide treatment and 24 h release, experiment performed twice with similar results. C Western blot of the nuclear-enriched fraction from Hs 578T cells of IMPDH2 and IgG immunoprecipitation showing IMPDH2, PARP1 and TOP2A, experiment performed twice with similar results. Western blot of MDA-MB-231 cells WT ( D ) and KO ( E ) showing full PARP1 and apoptotic and necrotic PARP1 cleaved, experiment performed twice; tubulin was used as a loading control. F Quantification of nuclear NAD+ as YFP/mCherry ratio across cell cycle phases in WT and KO cells cultured in the presence of guanosine 400 μM (WT: n G1 = 2316, n S = 1348, n G2/M = 1761; KO: n G1 = 2747, n S = 1359, n G2/M = 1282; outliers removed, 3 SD; unpaired two-tailed Wilcoxon test). Measurement of total cell death ( G ) and early apoptosis, late apoptosis/necrosis ( H ) in MDA-MB-231 WT and KO cells without guanosine supplementation for 96 h (WT: n = 6; KO: n = 6; unpaired two-tailed t-test); mean values +/− SD. Quantification of total cytosolic cleaved Casp3 Spot Area ( I ) (nWT= 79460, nKO= 7453; outliers removed, 3 SD; unpaired two-tailed Wilcoxon test), PARP1 cytosolic intensity ( J ) (nWT= 41640 nKO= 2297; outliers removed, 3 SD; unpaired two-tailed Wilcoxon test) and nuclear poly/mono ADP-ribose signal intensity ( K ) ( n WT = 32600, n KO = 364; unpaired two-tailed Wilcoxon test) in MDA-MB 231 WT and KO cells without guanosine supplementation for 96 h. Immunofluorescence of PARP1 representative pictures (magenta) and DAPI (blue) in DMSO control and 10 μM etoposide treatment after 24 h of release; non-confocal mode, scale bar 15μm ( L ) and number of PARP1 spots quantification in MDA-MB-231 cells (DMSO, n = 3748; n 1μM = 3165; n 2.5 μM = 3763; n 5μM = 2898; n 10μM = 3740; unpaired two-tailed Wilcoxon test) ( M ). All box plots indicate the median value (central line), interquartile range IQR (box boundaries), and up to 1.5*IQR beyond the box boundaries (whiskers). Source data are provided as a Source Data file.

Article Snippet: Protein interactors of TOP2A and PARP1 were derived from the OpenCell project .

Techniques: Western Blot, Immunoprecipitation, Control, Cell Culture, Two Tailed Test, Immunofluorescence

Journal: Nature Communications

Article Title: Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

doi: 10.1038/s41467-024-53877-z

Figure Lengend Snippet: A , B Western blot analysis of PARP1 fragmentation in WT, KO-WT, and KO-NLS treated with DMSO or etoposide without release and after 24 h release (experiments performed twice). Tubulin is used as loading control. Immunofluorescence pictures of IMPDH2 (green), DAPI (blue), and PARP1 (magenta) from DMSO and 10 μM 24 h etoposide treatment, scale bar 15μm ( C ), and quantification of cytosolic PARP1 spots in KO-WT and KO-NLS cells normalized by DMSO (DMSO, nKO-WT = 75011; nKO-NLS = 125677, n10μM KO-WT = 45449; n10μM KO-NLS = 75939). Unpaired two-tailed Wilcoxon test, ( n = 3 and 3 technical replicates per condition) ( D ). Immunofluorescence pictures of Phalloidin (red), cleaved Caspase3 (yellow) and DAPI (blue) from DMSO and 10μM 24 h etoposide treatment, scale bar 15 μm ( E ), and quantification of total cytosolic cleaved Caspase spot area in KO-WT and KO-NLS cells (DMSO, nKO-WT = 50568; nKO-NLS = 82455, n10μM KO-WT = 447736; n10μM KO-NLS = 80561). Unpaired two-tailed Wilcoxon test, ( n = 3 and 3 technical replicates per condition) ( F ). G Quantification of nuclear poly/mono ADP-ribose signal intensity in MDA-MB-231 WT, KO, KO-WT, KO-NLS cells in the absence of guanosine supplementation for 96 h (nWT = 32590, nKO= 4575, nKO-WT = 3798, nKO-NLS = 15868) ( n = 3 biological replicates, unpaired two-tailed Wilcoxon test). H Quantification of nuclear poly/mono ADP-ribose signal intensity for a titration with increasing NAD + concentrations in KO-WT and KO-NLS cells (nKO-NLS = 15868, n0 = 10376, n12.25 = 2194, n100 = 2965, nKO-WT = 3798; unpaired two-tailed Wilcoxon test); outliers removed, and constantly added to improve visualization. Quantification of intranuclear NAD + as YFP/mCherry ratio for KO-WT and KO-NLS cells with representative images (scale bar 15μm) after DMSO or 10 μM etoposide treatment for 2 h ( I ) and 24 h ( J ). Two-way Anova with either Tukey’s or Sidak multiple comparisons depending on intra or inter-comparison. For each timepoint 3 biological replicates were performed for the DMSO condition and 5 biological replicates for the ETO condition. All box plots indicate median value (central line), interquartile range IQR (box boundaries), and up to 1.5*IQR beyond the box boundaries (whiskers). Source data are provided as a Source Data file.

Article Snippet: Protein interactors of TOP2A and PARP1 were derived from the OpenCell project .

Techniques: Western Blot, Control, Immunofluorescence, Two Tailed Test, Titration, Comparison